Brassica plants yielding oils with a low alpha linolenic acid content

ABSTRACT

Brassica  plants producing oils with a low alpha-linolenic acid content and methods for producing such plants are described.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International Patent ApplicationNo. PCT/US2011/037864, filed May 25, 2011, which claims priority under35 U.S.C. §119(e) to U.S. Provisional Application Ser. No. 61/348,121,filed May 25, 2010.

TECHNICAL FIELD

This invention relates to Brassica plants, and more particularly,Brassica plants having a modified allele at a fatty acid desaturase 3Dlocus and/or a fatty acid desaturase 3E locus and yielding an oil with alow alpha linolenic acid content in combination with a typical, mid, orhigh oleic acid content.

BACKGROUND

Canola oil contains a relatively high (8%-10%) alpha-linolenic acid(ALA) content. This trienoic fatty acid is unstable and easily oxidizedduring cooking, which in turn creates off-flavors of the oil. It alsodevelops off odors and rancid flavors during storage (Hawrysh, 1990,Stability of canola oil, Chapter 7, pp. 99-122 In: F. Shahidi, ed.Canola and Rapeseed: Production, Chemistry, Nutrition, and ProcessingTechnology, Van Nostrand Reinhold, N.Y.). Reducing the ALA content levelby hydrogenation increases oxidative stability of the oil. However,hydrogenation results in the production of trans fatty acids, whichincreases the risk for coronary heart disease when consumed. Although anoil's oxidative stability is not determined solely by fatty acidprofile, a decrease in the ALA content of canola oils generally improvesthe stability of the oils.

SUMMARY

This document is based on the discovery of a modified fad3D allele and amodified fad3E allele, and use of such alleles in Brassica plants tocontrol ALA content. As described herein, Brassica plants containingsuch a modified fad3D allele and modified fad3E allele can produce oilswith a low ALA content (i.e. 1.5% or less ALA). Such Brassica plantsalso can include other modified fatty acid desaturase alleles (e.g.,fad2 or fad3), fatty acyl-acyl carrier protein thioesterase A2 (fatA2),and/or fatty acyl-acyl carrier protein thioesterase B (fatB) alleles totailor the oleic acid and total saturated fatty acid content to thedesired end use of the oil. Brassica plants described herein areparticularly useful for producing canola oils for certain foodapplications as the plants are not genetically modified.

In one aspect, this document features a Brassica plant (e.g., Brassicanapus, Brassica juncea, or Brassica rapa plant), progeny, and seeds ofthe plant that include a modified allele at a fatty acid desaturase 3D(fad3D) locus and/or a fatty acid desaturase 3E (fad3E) locus, whereinthe modified allele results in the production of a FAD3D and/or FAD3Epolypeptide having reduced desaturase activity relative to acorresponding wild-type polypeptide. The fad3E modified allele caninclude a nucleic acid encoding a truncated FAD3E polypeptide, a nucleicacid encoding a FAD3E polypeptide having a non-conservative substitutionof a residue affecting substrate specificity, or a nucleic acid encodinga FAD3E polypeptide having a non-conservative substitution of a residueaffecting catalytic activity. In some embodiments, the fad3E modifiedallele includes a mutation in a splice donor site. A modified fad3Eallele can include a nucleotide sequence having at least 95% sequenceidentity to the nucleotide sequence set forth in SEQ ID NO:1. The fad3Dmodified allele can include a nucleic acid encoding a truncated FAD3Dpolypeptide, a nucleic acid having a deletion of an exon or a portionthereof (e.g., a deletion within exon 1 of the nucleic acid). In someembodiments, the fad3D modified allele includes a nucleotide sequencehaving at least 95% sequence identity to the nucleic acid sequence setforth in SEQ ID NO:32. A plant can include fad3E and fad3D modifiedalleles. The fad3E and fad3D modified alleles can be mutant alleles. Aplant can be an F₁ hybrid.

Any of the plants described herein further can include a modified alleleat a fad3A locus and/or a modified allele at a fad3B locus. The fad3Aand/or fad3B modified alleles can be mutant alleles. For example, afad3A modified allele can be selected from the group consisting of a) anucleic acid encoding a FAD3A polypeptide having a cysteine substitutedfor arginine at position 275 and b) a nucleic acid encoding a truncatedFAD3A polypeptide. A fad3B modified allele can be selected from thegroup consisting of a) a nucleic acid having a mutation in anexon-intron splice site recognition sequence and b) a nucleic acidencoding a truncated FAD3B polypeptide. Such plants can produce seedsyielding an oil having an ALA content of 0.6 to 1.5%.

Plants described herein can produce seeds yielding an oil having astearic acid content of 2.5 to 6%.

Any of the plants described herein further can include a modified alleleat a delta-12 fatty acid desaturase (fad2) locus. The fad2 modifiedallele can include a nucleic acid encoding a FAD2 polypeptide having alysine substituted for glutamic acid in a His-Glu-Cys-Gly-His motif (SEQID NO:26). The fad2 modified allele comprising a nucleic acid encoding aFAD2 polypeptide having a glutamic acid substituted for glycine in theDRDYGILNKV motif (SEQ ID NO:28) or a histidine substituted for leucinein a KYLNNP motif (SEQ ID NO:27).

Any of the plants described herein further can include a modified alleleat two different fad2 loci. One fad2 modified allele can include anucleic acid encoding a FAD2 polypeptide having a lysine substituted forglutamic acid in a His-Glu-Cys-Gly-His motif (SEQ ID NO:26). One fad2modified allele can include a nucleic acid encoding a FAD2 polypeptidehaving a glutamic acid substituted for glycine in the DRDYGILNKV motif(SEQ ID NO:28) or a histidine substituted for leucine in a KYLNNP motif(SEQ ID NO:27).

Any of the plants described herein further can include a modified alleleat a fatty acyl-acyl-ACP thioesterase A2 (fatA2) locus and/or a fattyacyl-acyl-ACP thioesterase B (fatB) locus. The fatA2 and/or fatBmodified alleles can be mutant alleles. A fatA2 modified allele resultsin the production of a FATA2 polypeptide having reduced thioesteraseactivity relative to a corresponding wild-type FATA2 polypeptide. ThefatA2 modified allele can include a nucleic acid encoding a FATA2polypeptide having a mutation in a region (SEQ ID NO:11) correspondingto amino acids 242 to 277 of the FATA2 polypeptide. The FATA2polypeptide can include a substitution of a leucine residue for prolineat position 255. A fatB modified allele results in the production of aFATB polypeptide having reduced thioesterase activity relative to acorresponding wild-type FATB polypeptide. A plant can include modifiedalleles at four different fatB loci. At least one of the fatB modifiedalleles can include a nucleic acid encoding a truncated FATBpolypeptide. For example, a truncated FATB polypeptide can include anucleotide sequence selected from the group consisting of: SEQ ID NO:22SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25.

In another aspect, this document features a method of producing an oil.The method includes crushing seeds produced from at least one Brassicaplant described herein; and extracting oil from the crushed seeds,wherein the oil has, after refining, bleaching, and deodorizing, an ALAcontent of 0.6 to 1.5%. The oil further can have a stearic acid contentof 2.5 to 6.0%.

This document also features a method for making a Brassica progenyplant. The method includes crossing one or more first Brassica parentplants comprising a modified allele at a fad3E locus and/or a fad3Dlocus and one or more second Brassica parent plants comprising amodified allele at a different fad3 locus, wherein each modified alleleresults in the production of a FAD3 polypeptide having reduceddesaturase activity relative to a corresponding wild-type FAD3polypeptide; and selecting, for one to five generations, for progenyplants having a modified allele at the fad3E locus and/or fad3D locus,and the modified allele at the different fad3 locus, thereby obtainingthe Brassica plant.

In another aspect, this document features a method for making a Brassicaplant. The method includes obtaining one or more first Brassica parentplants comprising a modified allele at a fad3E locus and/or modifiedallele at a fad3D locus, wherein the fad3E or fad3D modified alleleresults in the production of a FAD3E or FAD3D polypeptide having reduceddesaturase activity relative to a corresponding wild-type FAD3polypeptide; obtaining one or more second Brassica parent plantscomprising a modified allele at a fad2 locus, the fad2 modified allelecomprising a nucleic acid encoding a FAD2 polypeptide having a lysinesubstituted for glycine in a His-Glu-Cys-Gly-His motif (SEQ ID NO:26);crossing the one or more first Brassica parent plants and the one ormore second Brassica parent plants; and selecting, for one to fivegenerations, for progeny plants having the modified allele at the fad3Elocus and/or fad3D locus, and the modified allele at the fad2 locusthereby obtaining the Brassica plant. The first Brassica parent plantcan include a modified allele at three different fad3 loci (e.g., fad3D,fad3A and fad3B).

In another aspect, this document features a method for making a Brassicaplant. The method includes obtaining one or more first Brassica parentplants comprising a modified allele at a fad3E locus and/or fad3D locus,wherein the fad3E or said fad3D modified allele results in theproduction of a FAD3E or FAD3D polypeptide having reduced desaturaseactivity relative to a corresponding wild-type FAD3 polypeptide;obtaining one or more second Brassica parent plants comprising amodified allele at a fatA2 locus, the fatA2 modified allele comprising anucleic acid encoding a FATA2 polypeptide having a mutation in a region(SEQ ID NO:11) corresponding to amino acids 242 to 277 of the FADA2polypeptide; crossing the one or more first Brassica parent plants andthe one or more second Brassica parent plants; and selecting, for one tofive generations, for progeny plants having the modified allele at thefad3E locus and/or the fad3D locus, and the modified allele at the fatA2locus thereby obtaining the Brassica plant. The first Brassica parentplant further can include a modified allele at a fad2 locus, a modifiedallele at a fad3A locus, and a modified allele at a fad3B locus, whereinthe fad2 modified allele comprising a nucleic acid encoding a FAD2polypeptide having a lysine substituted for glutamic acid in aHis-Glu-Cys-Gly-His motif (SEQ ID N:26), the fad3A modified allelecomprising a nucleic acid encoding a FAD3A polypeptide having a cysteinesubstituted for arginine at position 275, and the fad3B modified allelecomprising a fad3B nucleic acid sequence having a mutation in anexon-intron splice site recognition sequence.

This document also features a method for making a Brassica plant. Themethod includes obtaining one or more first Brassica parent plantscomprising a modified allele at a fad3E locus or a fad3D locus, whereinthe fad3E or fad3D modified allele results in the production of a FAD3Eor FAD3D polypeptide having reduced desaturase activity relative to acorresponding wild-type FAD3 polypeptide; obtaining one or more secondBrassica parent plants comprising at least one modified allele at a fatBlocus, wherein the fatB modified allele results in the production of aFATB polypeptide having reduced thioesterase activity relative to acorresponding wild-type FATB polypeptide; crossing the one or more firstBrassica parent plants and the one or more second Brassica parentplants; and selecting, for one to five generations, for progeny plantshaving the modified allele at the fad3E locus and/or fad3D locus, andthe at least one modified fatB allele at the fatB locus, therebyobtaining the Brassica plant. The one or more second Brassica plants caninclude modified alleles at four different fatB loci. At least one ofthe fatB modified alleles can include a nucleic acid encoding atruncated FATB polypeptide.

In another aspect, this document features seeds of a Brassica plantcomprising a modified allele at a fad3E locus and/or a modified alleleat a fad3D locus. The fad3E modified allele can include a nucleic acidhaving a mutation in a splice donor site. The fad3D modified allele caninclude a nucleic acid having a deletion of a portion of exon 1. Theseeds can yield an oil having an ALA content of 0.6% to 1.5%. The seedscan be F₂ seeds. The Brassica plant further can include modified allelesat fad3A and/or fad3B loci. The Brassica plant further can include amodified allele at a fad2 locus. The Brassica plant further can includea modified allele at a fatB locus. The Brassica plant further caninclude a modified allele at a fatA2 locus. The Brassica plant furthercan include modified alleles at fad3A, fad3B, fad2, fatB, and fatA2loci.

In yet another aspect, this document features a plant cell of a plantdescribed herein, wherein the plant cell includes one or more of themodified alleles.

This document also features an isolated nucleic acid that includes anucleic acid sequence selected from the group consisting of i) thenucleic acid sequence set forth in SEQ ID NO:1; ii) the nucleic acidsequence set forth in SEQ ID NO:32; iii) the complement of the nucleicacid sequence set forth in i) or ii); and iv) a nucleic acid fragment ofi), ii), or iii) that is at least 50 nucleotides in length anddistinguishes a mutant fad3D or fad3E allele from a wild-type fad3D orfad3E allele.

In another aspect, this document features a method of making a plantline. The method includes providing a population of plants; identifyingone or more plants in the population containing a modified allele at afad3E locus and/or a fad3D locus, wherein the modified allele results inthe production of a FAD3E or FAD3D polypeptide having reduced desaturaseactivity relative to a corresponding wild-type FAD3 polypeptide;crossing one or more of the identified plants with itself or a differentplant to produce seed; crossing at least one progeny plant grown fromthe seed with itself or a different plant; and repeating the crossingsteps for an additional 0-5 generations to make the plant line, whereinthe modified allele at the fad3E locus and/or the fad3D locus is presentin the plant line.

This document also features Brassica napus seed designated 1904 andrepresented by American Type Culture Collection (ATCC) Accession No.PTA-11273, as well as progeny of the seed designated 1904 andrepresented by ATCC Accession No. PTA-11273.

This document also features Brassica napus seed designated 2558 andrepresented by American Type Culture Collection (ATCC) Accession No.PTA-11274, as well as progeny of the seed designated 2558 andrepresented by ATCC Accession No. PTA-11274.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All numbers expressingquantities of ingredients, properties such as molecular weight,percentages, reaction conditions, and so forth used in the specificationand claims are to be understood as being modified by the term “about.”Accordingly, unless indicated to the contrary, the numerical parametersset forth are approximations that may depend upon the desired propertiessought.

Although methods and materials similar or equivalent to those describedherein can be used to practice the invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is an alignment of the BnFad3E sequences from 1904, IMC201, andBrFad3E (SEQ ID NOs:1, 2, and 3, respectively). The BnFad3E-2 SNP thatcorrelates with the low ALA (C18:3) content in the 1904 mutant line ishighlighted with a black box at position 1851 of this alignment. At theposition 1756 in SEQ ID NO:1 (1904 BnFad3E-2.seq), a single nucleotidemutation (G to A) is shown, which is located in a splice donor site (seeFIG. 2). The start codon (ATG) is underlined at position 94 of thissequence alignment and the stop codon in BrFad3E (TAA) is at position3828.

FIG. 2 is an alignment of the nucleotide sequence of the exon 3, intron3 border of the BnFad3A, BnFad3B, BnFad3E genes from IMC201, IMC02,Westar, 1904, 2558, and 95CB504, and the BrFad3E gene from Brassica rapa(world wide web at brassica-rapa.org) showing the single nucleotidemutation (G to A) in BnFad3E-2 from the 1904 line. See SEQ ID NOs:4-8.This mutation (G to A) is located in the last nucleotide of exon 3 ofBnFad3E. Intron 3 of the BnFad3E starts from the sequence GT (see SEQ IDNO:8).

FIG. 3 is an alignment of the amino acid sequences of FAD3E polypeptidesfrom B. napus and B. rapa, and FAD3 from Arabidopsis thaliana. See SEQID NOs:29, 30, and 31.

FIG. 4 is an alignment of the BnFad3D sequences from 1904 (SEQ ID NO:32)and IMC201 and 95CB504 (SEQ ID NO:33) showing a DNA deletion in the 1904BnFad3D starting at position 575 in this alignment, which includes aportion of exon 1 and intron 1. The start codon (ATG) is at position441.

FIG. 5 is the amino acid sequence of the FAD3D polypeptide (SEQ IDNO:34). In line 1904, the FAD3D polypeptide is truncated after aminoacid 64.

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION

In general, this document provides Brassica plants, including B. napus,B. juncea, and B. rapa species of Brassica, that yield seeds producingoils having a low ALA content (i.e., 1.6% or less). Canola oil producedfrom seeds having a low ALA content tends to exhibit increased stability(e.g., oxidative stability and/or flavor stability) and a usefulnutritional profile, and can be used for many food applicationsincluding as a frying oil.

In some embodiments, plants described herein yield seeds producing oilshaving a low ALA content in combination with low total saturated fattyacids (i.e., 6% or less) or very low total saturated fatty acids (i.e.,having 3.6% or less). As used herein, total saturated fatty acid contentrefers to the total of myristic acid (C14:0), palmitic acid (C16:0),stearic acid (C18:0), arachidic acid (C20:0), behenic acid (C22:0), andlignoceric acid (C24:0). For example, Brassica plants described hereincan produce oils having a low ALA content and a total saturated fattyacid content of 2.5 to 6.0%, 3 to 5%, 3 to 4.5%, 3.25 to 3.75%, 3.0 to3.5%, 3.4 to 3.7%, 3.6 to 5%, 4 to 5.5%, 4 to 5%, or 4.25 to 5.25%. Oilshaving a low ALA content and a low or very low total saturated fattyacid content have improved oxidative stability and nutritional qualityand can help consumers reduce their intake of saturated fatty acids.

In some embodiments, Brassica plants yield seed oils having a low ALAcontent in combination with a typical (60%-70%), mid (70.1%-80%), orhigh (>80%) oleic acid content. In some embodiments, the total saturatedfatty acid content of such seed oils can be less than 6%. As such,Brassica plants can produce seed oils having a fatty acid contenttailored to the desired end use of the oil (e.g., frying or foodapplications). For example, Brassica plants can be produced that yieldseeds having a low ALA content (e.g., 1.5% or less), an oleic acidcontent of 60 to 70%, and a linoleic acid content of 17 to 24%. Canolaoils having such a fatty acid profile are particularly useful for fryingapplications due to the polyunsaturated fatty acid content, which is lowenough to have improved oxidative stability for frying yet high enoughto impart the desired fried flavor to the food being fried, and are animprovement over commodity type canola oils. The fatty acid content ofcommodity type canola oils may be on the order of is 6 to 8% totalsaturated fatty acids, 55 to 65% oleic acid, 20 to 30% linoleic acid,and 7 to 10% α-linolenic acid. See, e.g., Bailey's Industrial Oil andFat Products, Section 2.2, “Canola Oil” on pages 61-121 of Volume 2 (6thEdition, 2005).

In some embodiments, Brassica plants can be produced that yield seedshaving a low ALA content, mid-oleic acid content (e.g., 70.1 to 80%oleic acid) and a low total saturated fatty acid content (e.g., <6.0%).Canola oils having such a fatty acid profile have an oxidative stabilitythat is higher than oils with higher ALA and lower oleic acid contentsor commodity type canola oils, and are useful for coating applications(e.g., spray-coatings), formulating food products, or other applicationswhere shelf-life stability is desired. In addition, Brassica plants canbe produced that yield seeds having a low ALA content, high oleic acidcontent (e.g., 80.1 to 90% oleic acid) and a low total saturated fattyacid content (<6.0%). Canola oils having a low ALA, high oleic acid, andlow total saturated fatty acid content are particularly useful for foodapplications requiring high oxidative stability and a reduced saturatedfatty acid content.

Brassica Plants

Brassica plants described herein can have reduced levels of ALA (e.g.,8.0% or less) in the seed oil as a result of reduced activity of fattyacid desaturase (FAD) 3E (also known as delta-15 desaturase). Brassicaplants described herein also can have reduced levels of ALA (e.g., 3.0%or less, 2.8% or less, 2.6% or less) in the seed oil as a result ofreduced activity of FAD3D. FAD3 proteins are involved in the enzymaticconversion of linoleic acid to α-linolenic acid. Sequences of higherplant Fad3 genes are disclosed in Yadav et al., Plant Physiol.,103:467-476 (1993), WO 93/11245, and Arondel et al., Science,258:1353-1355 (1992). It is understood that throughout the disclosure,reference to “plant” or “plants” includes progeny, i.e., descendants ofa particular plant or plant line, as well as cells or tissues from theplant. Progeny of an instant plant include seeds formed on F₁, F₂, F₃,F₄ and subsequent generation plants, or seeds formed on BC₁, BC₂, BC₃,and subsequent generation plants. Seeds produced by a plant can be grownand then selfed (or outcrossed and selfed, or doubled through dihaploid)to obtain seeds homozygous for a modified allele. The term “allele” or“alleles” refers to one or more alternative forms of a gene at aparticular locus. As used herein, a “line” is a group of plants thatdisplay little or no genetic variation between individuals for at leastone trait. Such lines may be created by several generations ofself-pollination and selection, or vegetative propagation from a singleparent using tissue or cell culture techniques. As used herein, the term“variety” refers to a line which is used for commercial production, andincludes hybrid varieties and open-pollinated varieties.

Reduced activity, including absence of detectable desaturase activity,of FAD3E and/or FAD3D can be achieved by modifying an endogenous fad3Eor fad3D allele. An endogenous fad3E or fad3D allele can be modified by,for example, mutagenesis or by using homologous recombination to replacean endogenous plant gene with a variant containing one or more mutations(e.g., produced using site-directed mutagenesis). See, e.g., Townsend etal., Nature 459:442-445 (2009); Tovkach et al., Plant J., 57:747-757(2009); and Lloyd et al., Proc. Natl. Acad. Sci. USA, 102:2232-2237(2005). Similarly, for other genes discussed herein, the endogenousallele can be modified by mutagenesis or by using homologousrecombination to replace an endogenous gene with a variant. Modifiedalleles obtained through mutagenesis are encompassed by the term “mutantalleles” as that term is used herein.

Reduced desaturase activity, including absence of detectable activity,can be inferred from the decreased level of linolenic acid (product) andin some cases, increased level of linoleic acid (the substrate) in theplant compared with a corresponding control plant. Reduced activity alsocan be assessed by in vitro translation of the desaturase and assayingfor desaturase activity. See, for example, Goren and Fox, Protein ExprPurif. 62(2): 171-178 (2008).

Genetic mutations can be introduced within a population of seeds orregenerable plant tissue using one or more mutagenic agents. Suitablemutagenic agents include, for example, ethyl methane sulfonate (EMS),methyl N-nitrosoguanidine (MNNG), ethidium bromide, diepoxybutane,ionizing radiation, x-rays, UV rays and other mutagens known in the art.In some embodiments, a combination of mutagens, such as EMS and MNNG,can be used to induce mutagenesis. The treated population, or asubsequent generation of that population, can be screened for reduceddesaturase activity that results from the mutation, e.g., by determiningthe fatty acid profile of the population and comparing it to that of acorresponding non-mutagenized population. Mutations can be in anyportion of a gene, including coding sequence, exon sequence, intronsequence, and regulatory elements, that render the resulting geneproduct non-functional or with reduced activity. Suitable types ofmutations include, for example, insertions or deletions of nucleotides,and transitions or transversions in the wild-type coding sequence. Suchmutations can lead to deletion or insertion of amino acids, andconservative or non-conservative amino acid substitutions in thecorresponding gene product. In some embodiments, the mutation is adeletion of an exon or a portion thereof, resulting in the production ofa truncated polypeptide from either lack of or incorrect RNA splicing.In some embodiments, the mutation is a nonsense mutation, which resultsin the introduction of a stop codon (TGA, TAA, or TAG) and production ofa truncated polypeptide. The gene product of an allele having a stopcodon mutation typically lacks detectable desaturase activity. In someembodiments, the mutation is a splice site mutation which alters orabolishes the correct splicing of the pre-mRNA sequence, resulting in aprotein of different amino acid sequence than the wild type. Forexample, one or more exons may be skipped during RNA splicing, resultingin a protein lacking the amino acids encoded by the skipped exons.Alternatively, the reading frame may be altered by incorrect splicing,one or more introns may be retained, alternate splice donors oracceptors may be generated, or splicing may be initiated at an alternateposition, or alternative polyadenylation signals may be generated. Insome embodiments, more than one mutation or more than one type ofmutation is introduced. PCR can be used to amplify modified alleles ingenomic DNA from the plant or plant tissue, and the resultingamplification product can be isolated and sequenced to characterize thepolypeptide encoded by the modified allele. In some embodiments, RT-PCRcan be used to detect particular RNA transcripts.

Insertions, deletions, or substitutions of amino acids in a proteinsequence may, for example, disrupt the conformation of essentialalpha-helical or beta-pleated sheet regions of the resulting geneproduct. Amino acid insertions, deletions, or substitutions also candisrupt binding, alter substrate specificity, or disrupt catalytic sitesimportant for gene product activity. It is known in the art that theinsertion or deletion of a larger number of contiguous amino acids ismore likely to render the gene product non-functional, compared to asmaller number of inserted or deleted amino acids. Non-conservativeamino acid substitutions may replace an amino acid of one class with anamino acid of a different class. Non-conservative substitutions may makea substantial change in the charge or hydrophobicity of the geneproduct. Non-conservative amino acid substitutions may also make asubstantial change in the bulk of the residue side chain, e.g.,substituting an alanine residue for an isoleucine residue.

Examples of non-conservative substitutions include the substitution of abasic amino acid for a non-polar amino acid, or a polar amino acid foran acidic amino acid. Because there are only 20 amino acids encoded in agene, substitutions that result in reduced activity may be determined byroutine experimentation, incorporating amino acids of a different classin the region of the gene product targeted for mutation.

In some embodiments, a plant described herein contains a modified alleleat a fad3E locus. For example, a fad3E locus can include a nucleotidesequence having at least 90% (e.g., at least 91, 92, 93, 94, 95, 96, 97,98, or 99%) sequence identity to the nucleotide sequence set forth inSEQ ID NO:1. The nucleotide sequence set forth in SEQ ID NO:1 is arepresentative nucleotide sequence of the fad3E gene from B. napus line1904, which contains a single nucleotide mutation (G to A) in a splicedonor site. As used herein, the term “sequence identity” refers to thedegree of similarity between any given nucleic acid sequence and atarget nucleic acid sequence. The degree of similarity is represented aspercent sequence identity. Percent sequence identity is calculated bydetermining the number of matched positions in aligned nucleic acidsequences, dividing the number of matched positions by the total numberof aligned nucleotides, and multiplying by 100. A matched positionrefers to a position in which identical nucleotides occur at the sameposition in aligned nucleic acid sequences. Percent sequence identityalso can be determined for any amino acid sequence. Percent sequenceidentity can be determined using the BLAST 2 Sequences (Bl2seq) programfrom the stand-alone version of BLASTZ containing BLASTN version 2.0.14and BLASTP version 2.0.14. This stand-alone version of BLASTZ can beobtained from Fish & Richardson's web site (World Wide Web at “fr” dot“com” slash “blast”) or the U.S. government's National Center forBiotechnology Information web site (World Wide Web at “ncbi” dot “nlm”dot “nih” dot “gov”). Instructions explaining how to use the Bl2seqprogram can be found in the readme file accompanying BLASTZ.

Bl2seq performs a comparison between two sequences using either theBLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acidsequences, while BLASTP is used to compare amino acid sequences. Tocompare two nucleic acid sequences, the options are set as follows: -iis set to a file containing the first nucleic acid sequence to becompared (e.g., C:\seq1.txt); -j is set to a file containing the secondnucleic acid sequence to be compared (e.g., C:\seq2.txt); -p is set toblastn; -o is set to any desired file name (e.g., C:\output.txt); -q isset to −1; -r is set to 2; and all other options are left at theirdefault setting. The following command will generate an output filecontaining a comparison between two sequences: C:\Bl2seq -i c:\seq1.txt-j c:\seq2.txt -p blastn -o c:\output.txt -q -1-r 2. If the targetsequence shares homology with any portion of the identified sequence,then the designated output file will present those regions of homologyas aligned sequences. If the target sequence does not share homologywith any portion of the identified sequence, then the designated outputfile will not present aligned sequences.

Once aligned, a length is determined by counting the number ofconsecutive nucleotides from the target sequence presented in alignmentwith sequence from the identified sequence starting with any matchedposition and ending with any other matched position. A matched positionis any position where an identical nucleotide is presented in both thetarget and identified sequence. Gaps presented in the target sequenceare not counted since gaps are not nucleotides. Likewise, gaps presentedin the identified sequence are not counted since target sequencenucleotides are counted, not nucleotides from the identified sequence.

The percent identity over a particular length is determined by countingthe number of matched positions over that length and dividing thatnumber by the length followed by multiplying the resulting value by 100.For example, if (i) a 500-base nucleic acid target sequence is comparedto a subject nucleic acid sequence, (ii) the Bl2seq program presents 200bases from the target sequence aligned with a region of the subjectsequence where the first and last bases of that 200-base region arematches, and (iii) the number of matches over those 200 aligned bases is180, then the 500-base nucleic acid target sequence contains a length of200 and a sequence identity over that length of 90% (i.e., 180,200×100=90).

It will be appreciated that different regions within a single nucleicacid target sequence that aligns with an identified sequence can eachhave their own percent identity. It is noted that the percent identityvalue is rounded to the nearest tenth. For example, 78.11, 78.12, 78.13,and 78.14 are rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18,and 78.19 are rounded up to 78.2. It also is noted that the length valuewill always be an integer.

In some embodiments, a plant described herein contains a modified alleleat a fad3D locus. For example, a fad3D locus can include a nucleotidesequence having at least 90% (e.g., at least 91, 92, 93, 94, 95, 96, 97,98, or 99%) sequence identity to the nucleotide sequence set forth inSEQ ID NO:32. The nucleotide sequence set forth in SEQ ID NO:32 is arepresentative nucleotide sequence of the fad3D gene from B. napus line1904, which contains a deletion of 164 nucleotides from exon 1. In B.napus line IMC201 and 95CB504, exon 1 starts at position 441 and ends atposition 739. See, e.g., FIG. 4.

In some embodiments, a Brassica plant contains a modified fad3E alleleand a modified fad3D allele. A modified fad3E and a modified fad3Dallele may be combined in a plant by making a genetic cross betweenmodified lines. For example, a plant having a modified allele at a fad3Elocus can be crossed or mated with a second plant having a modifiedallele at a fad3D locus. Seeds produced from the cross are planted andthe resulting plants are selfed in order to obtain progeny seeds. Theseprogeny seeds can be screened in order to identify those seeds carryingboth modified alleles. In some embodiments, progeny are selected overmultiple generations (e.g., 2 to 5 generations) to obtain plants havingmodified fad3E and fad3D alleles. In some embodiments, a line havingboth fad3E and fad3D modified alleles is used to introgress anindividual modified allele into a different line or to introgress bothmodified alleles into a different line.

In some embodiments, a Brassica plant contains a modified fad3E alleleor a modified fad3D allele, and optionally one or more modified allelesat fad3 (e.g. fad3A and/or fad3B), fatA2, fatB, and fad2 loci. In someembodiments, a Brassica plant contains a modified fad3E allele and amodified fad3D allele, and optionally one or more modified alleles atfad3 (e.g., fad3A and/or fad3B), fatA2, fatB, and fad2 loci. Forexample, a Brassica plant can contain a modified fad3E allele, amodified fad3D allele, and one or more other modified fad3 alleles. Forexample, in addition to a modified fad3E and fad3D allele, Brassicaplants can contain the mutation from the APOLLO or STELLAR B. napusvariety that confers low linolenic acid. The STELLAR and APOLLOvarieties were developed at the University of Manitoba (Manitoba,Canada). In some embodiments, the disclosed plants contain the fad3Aand/or fad3B mutation from IMC02 that confer a low linolenic acidphenotype. IMC02 contains a mutation in both the fad3A and fad3B genes.The fad3A gene contains a C to T mutation at position 2565, numberedfrom the ATG in genomic DNA, resulting in the substitution of a cysteinefor arginine at position 275 of the encoded FAD3A polypeptide. The fad3Bgene contains a G to A mutation at position 3053 from ATG in genomicDNA, located in the exon-intron splice site recognition sequence. IMC02was obtained from a cross of IMC01 x Westar. See Example 3 of U.S. Pat.No. 5,750,827. IMC01 was deposited with the American Type CultureCollection (ATCC) under Accession No. 40579. IMC02 was deposited withthe ATCC under Accession No. PTA-6221. Other examples of fad3 mutationsinclude nonsense mutations in fad3A and fad3B sequences. See, Example 4.For example, the mutant fad3A sequence set forth in SEQ ID NO:9 containsa mutation at position 102, resulting in a codon change from TGG to TGAand production of a truncated FAD3A polypeptide. The mutant fad3Bsequence set forth in SEQ ID NO:10 contains a mutation at position 206,resulting in a codon change from TGG to TAG and production of atruncated FAD3B polypeptide.

Two or more different modified fad3 alleles may be combined in a plantby making a genetic cross between modified lines. For example, a planthaving a modified allele at a fad3E locus and/or a fad3D locus can becrossed or mated with a second plant having a modified allele at a fad3Aor fad3B locus. Seeds produced from the cross are planted and theresulting plants are selfed in order to obtain progeny seeds. Theseprogeny seeds can be screened in order to identify those seeds carryingboth modified alleles. In some embodiments, progeny are selected overmultiple generations (e.g., 2 to 5 generations) to obtain plants havingmodified alleles at two different fad3 loci.

Brassica plants having a modified allele at a fad3E locus or fad3D locusalso can include modified alleles controlling fatty acyl-ACPthioesterase A2 (fatA2) and/or fatty acyl-ACP thioesterase B (fatB) totailor the total saturated fatty acid content to the end use of the oil.Fatty acyl-ACP thioesterases hydrolyze acyl-ACPs in the chloroplast torelease the newly synthesized fatty acid from ACP, effectively removingit from further chain elongation in the plastid. The free fatty acid canthen leave the plastid, become bound to CoenzymeA (CoA) and enter theKennedy pathway in the endoplasmic reticulum (ER) for triacylglycerol(TAG) biosynthesis. Members of the FATA family prefer oleoyl (C18:1) ACPsubstrates with minor activity towards 18:0 and 16:0-ACPs, while membersof the FATB family hydrolyze primarily saturated acyl-ACPs between 8 and18 carbons in length. See Jones et al., Plant Cell 7:359-371 (1995);Ginalski and Rhchlewski, Nucleic Acids Res 31:3291-3292 (2003); andVoelker T in Genetic Engineering (Setlow, J K, ed) Vol 18, 111-133,Plenum Publishing Corp., New York (2003).

Reduced activity of FATA2 and/or FATB, including absence of detectableactivity, can be inferred from the decreased level of saturated fattyacids in the seed oil compared with seed oil from a correspondingcontrol plant. Reduced activity also can be assessed in plant extractsusing assays for fatty acyl-ACP hydrolysis. See, for example,Bonaventure et al., Plant Cell 15:1020-1033 (2003); and Eccleston andOhlrogge, Plant Cell 10:613-622 (1998).

In some embodiments, in addition to a modified allele at a fad3E locusand/or a fad3D locus, and optionally one or more other modified fad3loci, a Brassica plant contains a modified allele at a fatA2 locus,wherein the modified allele results in the production of a FATA2polypeptide having reduced thioesterase activity relative to acorresponding wild-type FATA2 polypeptide. For example, the modifiedfatA2 allele can include a nucleic acid that encodes a FATA2 polypeptidehaving a non-conservative substitution within a helix/4-stranded sheet(4HBT) domain (also referred to as a hot-dog domain) or non-conservativesubstitution of a residue affecting catalytic activity or substratespecificity. For example, a Brassica plant can contain a modified allelethat includes a nucleic acid encoding a FATA2b polypeptide having asubstitution in a region (SEQ ID NO:11) of the polypeptide correspondingto residues 242 to 277 of the FATA2 polypeptide (as numbered based onthe alignment to the Arabidopsis thaliana FATA2 polypeptide set forth inGenBank Accession No. NP_(—)193041.1, protein (SEQ ID NO:12); GenBankAccession No. NM_(—)117374, mRNA). This region of FATA2 is highlyconserved in Arabidopsis and Brassica. In addition, many residues inthis region are conserved between FATA and FATB, including the asparticacid at position 259, asparagine at position 263, histidine at position265, valine at position 266, asparagine at position 268, and tyrosine atposition 271 (as numbered based on the alignment to SEQ ID NO:12). Theasparagine at position 263 and histidine at position 265 are part of thecatalytic triad, and the arginine at position 256 is involved indetermining substrate specificity. See also Mayer and Shanklin, BMCPlant Biology 7:1-11 (2007). SEQ ID NO:13 sets forth the predicted aminoacid sequence of the Brassica FATA2b polypeptide encoded by exons 2-6,and corresponding to residues 121 to 343 of the A. thaliana sequence setforth in SEQ ID NO:12. For example, the FATA2 polypeptide can have asubstitution of a leucine residue for proline at the positioncorresponding to position 255 of the Arabidopsis FATA2 polypeptide(i.e., position 14 of SEQ ID NO:11 or position 135 of SEQ ID NO: 13).The proline in the B. napus sequence corresponding to position 255 inArabidopsis is conserved among B. napus, B. rapa, B. juncea, Zea mays,Sorghum bicolor, Oryza sativa Indica (rice), Triticum aestivum, Glycinemax, Jatropha (tree species), Carthamus tinctorius, Cuphea hookeriana,Iris tectorum, Perilla frutescens, Helianthus annuus, Garciniamangostana, Picea sitchensis, Physcomitrella patens subsp. Patens,Elaeis guineensis, mitis vinifera, Elaeis oleifera, Camellia oleifera,Arachis hypogaea, Capsicum annuum, Cuphea hookeriana, Populustrichocarpa, and Diploknema butyracea. The mutation at position 255 isassociated with a low total saturated fatty acid phenotype, low stearicacid phenotype, low arachidic acid phenotype, and an increasedeicosenoic acid phenotype. The stearic acid content phenotype isnegatively correlated with the eicosenoic acid phenotype. See, U.S.Provisional Application Nos. 61/287,985 and 61/295,049.

In some embodiments, the modified allele at a fatA2 locus includes anucleotide sequence having at least 90% (e.g., at least 91, 92, 93, 94,95, 96, 97, 98, or 99%) sequence identity to the nucleotide sequence setforth in SEQ ID NO:14 or SEQ ID NO:15. The nucleotide sequences setforth in SEQ ID NOs:14 and 15 are representative nucleotide sequencesfrom the mutant fatA2b gene from B. napus line 15.24. See, U.S.Provisional Application Nos. 61/287,985 and 61/295,049.

In some embodiments, a Brassica plant contains a modified allele at afatB locus, wherein the modified allele results in the production of aFATB polypeptide having reduced thioesterase activity relative to acorresponding wild-type FATB polypeptide. In some embodiments, aBrassica plant contains modified alleles at two or more different fatBloci. In some embodiments, a Brassica plant contains modified alleles atthree different fatB loci or contains modified alleles at four differentfatB loci. Brassica napus contains 6 different FATB isoforms (i.e.,different forms of the FATB polypeptide at different loci), which arecalled isoforms 1-6 herein. SEQ ID NOs:16-21 set forth the nucleotidesequences encoding FATB isoforms 1-6, respectively, of Brassica napus.The nucleotide sequences set forth in SEQ ID NOs:16-21 have 82% to 95%sequence identity as measured by the ClustalW algorithm (version 1.83,default parameters). See Chenna et al., Nucleic Acids Res.,31(13):3497-500 (2003).

For example, in addition to a modified allele at a fad3E locus and afad3D locus, a Brassica plant can have a mutation in a nucleotidesequence encoding FATB isoform 1, isoform 2, isoform 3, isoform 4,isoform 5, or isoform 6. In some embodiments, a plant can have amutation in a nucleotide sequence encoding FAD3E and can have mutationin a nucleotide sequence encoding 2 or more FATB isoforms, e.g., FATBisoforms 1 and 2; 1 and 3; 1 and 4; 1 and 5; 1 and 6; 2 and 3; 2 and 4;2 and 5; 2 and 6; 3 and 4; 3 and 5; 3 and 6; 4 and 5; 4 and 6; 5 and 6;1, 2, and 3; 1, 2, and 4; 1, 2, and 5; 1, 2, and 6; 1, 3, and 4; 1, 3,and 5; 1, 3, and 6; 1, 4, and 5; 1, 4, and 6; 1, 5, and 6; 2, 3, and 4;2, 3, and 5; 2, 3, and 6; 2, 4, and 5; 2, 4, and 6; 1, 5, and 6; 3, 4,and 5; 3, 4, and 6; 3, 5, and 6; 4, 5, and 6; 1, 2, 3, and 4; 1, 2, 3,and 5; 1, 2, 3, and 6; 1, 2, 4, and 5; 1, 2, 4, and 6; 1, 2, 5, and 6;1, 3, 4 and 5; 1, 3, 4, and 6; 1, 3, 5, and 6; 1, 4, 5, and 6; 2, 3, 4,and 5; 2, 3, 4 and 6; 2, 3, 5, and 6; 2, 4, 5, and 6; or 3, 4, 5, and 6.In some embodiments, a Brassica plant can have a mutation in anucleotide sequence encoding a FAD3E polypeptide and can have a mutationin nucleotide sequences encoding FATB isoforms 1, 2, and 3; 1, 2, and 4;1, 3, and 4; 2, 3, and 4; or 1, 2, 3, and 4. In some embodiments, amutation in a FATB isoform results in deletion of a 4HBT domain or aportion thereof of a FATB polypeptide. FATB polypeptides typicallycontain a tandem repeat of the 4HBT domain, where the N-terminal 4HBTdomain contains residues affecting substrate specificity (e.g., twoconserved methionines, a conserved lysine, a conserved valine, and aconserved serine) and the C-terminal 4HBT domain contains residuesaffecting catalytic activity (e.g., a catalytic triad of a conservedasparagine, a conserved histidine, and a conserved cysteine) andsubstrate specificity (e.g., a conserved tryptophan). See Mayer andShanklin, J. Biol. Chem. 280:3621-3627 (2005). In some embodiments, themutation in a nucleotide sequence encoding FATB results in anon-conservative substitution of a residue in a 4HBT domain or a residueaffecting substrate specificity. In some embodiments, the mutation in anucleotide sequence encoding FATB is a splice site mutation. In someembodiment, the mutation in a nucleotide sequence encoding FATB is anonsense mutation in which a premature stop codon (TGA, TAA, or TAG) isintroduced, resulting in the production of a truncated polypeptide.

SEQ ID NOs:22-25 set forth the nucleotide sequences encoding fatBisoforms 1-4, respectively, and containing exemplary nonsense mutationsthat result in truncated FATB polypeptides. SEQ ID NO:22 is thenucleotide sequence of isoform 1 having a mutation at position 154,which changes the codon from CAG to TAG. SEQ ID NO:23 is the nucleotidesequence of isoform 2 having a mutation at position 695, which changesthe codon from CAG to TAG. SEQ ID NO:24 is the nucleotide sequence ofisoform 3 having a mutation at position 276, which changes the codonfrom TGG to TGA. SEQ ID NO:25 is the nucleotide sequence of isoform 4having a mutation at position 336, which changes the codon from TGG toTGA. See also U.S. Provisional Application Nos. 61/287,985 and61/295,049.

Two or more different modified FATB alleles may be combined in a plantby making a genetic cross between modified lines. For example, a planthaving a modified allele at a FATB locus encoding isoform 1 can becrossed or mated with a second plant having a modified allele at a FATBlocus encoding isoform 2. Seeds produced from the cross are planted andthe resulting plants are selfed in order to obtain progeny seeds. Theseprogeny seeds can be screened in order to identify those seeds carryingboth modified alleles. In some embodiments, progeny are selected overmultiple generations (e.g., 2 to 5 generations) to obtain plants havingmodified alleles at two different FATB loci. Similarly, a plant havingmodified alleles at two or more different FATB isoforms can be crossedwith a second plant having modified alleles at two or more differentFATB alleles, and progeny seeds can be screened to identify those seedscarrying modified alleles at four or more different FATB loci. Again,progeny can be selected for multiple generations to obtain the desiredplant.

In some embodiments, a modified allele at a fad3E locus, a fad3D locus,a fatA2 locus and modified alleles at two or more (e.g., three or four)different fatB loci can be combined in a plant. For example, a planthaving a modified allele at a fad3E locus and a fad3D locus can becrossed or mated with a second plant having a modified allele at a fatA2locus. Seeds produced from the cross are planted and the resultingplants are selfed in order to obtain progeny seeds. These progeny seedscan be screened in order to identify those seeds carrying modifiedfad3E, fad3D, and fatA2 alleles. Progeny can be selected over multiplegenerations (e.g., 2 to 5 generations) to obtain plants having amodified allele at a fad3E locus, a modified allele at a fad3D locus,and a modified allele at a fatA2 locus. Furthermore, progeny identifiedas having a modified allele at a fad3E locus, a modified allele at afad3D locus, and a modified allele at a fatA2 locus can be crossed ormated with a second plant having modified alleles at two or moredifferent fatB loci. Seeds produced from the cross are planted and theresulting plants are selfed in order to obtain progeny seeds. Theseprogeny seeds can be screened in order to identify those seeds carryingmodified fad3E, fad3D, fatA2, and fatB alleles. Progeny can be selectedover multiple generations (e.g., 2 to 5 generations) to obtain plantshaving a modified allele at a fad3E locus, a modified allele at a fatA2locus, and two or more different fatB loci. Plants having a modifiedallele at a fad3E locus, a fad3D locus, a fatA2b locus, and modifiedalleles at three or four different fatB loci have a low ALA content,high oleic acid content, and a low total saturated fatty acid content.

Brassica plants described herein also can have decreased activity of adelta-12 desaturase, which is involved in the enzymatic conversion ofoleic acid to linoleic acid, to confer a mid or high oleic acid contentin the seed oil. Brassica plants can exhibit reduced activity ofdelta-12 desaturase (also known as FAD2) in combination with reducedactivity of FAD3E and optionally one or more of FAD3A, FAD3B, FATA2, andFATB. The sequences for the wild-type fad2 genes from B. napus (termedthe D form and the F form) are disclosed in WO 98/56239. A reduction indelta-12 desaturase activity, including absence of detectable activity,can be achieved by mutagenesis. Decreased delta-12 desaturase activitycan be inferred from the decrease level of linoleic acid (product) andincreased level of oleic acid (substrate) in the plant compared with acorresponding control plant. Non-limiting examples of suitable fad2mutations include the G to A mutation at nucleotide 316 within thefad2-D gene, which results in the substitution of a lysine residue forglutamic acid in a HECGH (SEQ ID NO:26) motif. Such a mutation is foundwithin the line IMC129, which has been deposited with the ATCC underAccession No. 40811. Another suitable fad2 mutation can be the T to Amutation at nucleotide 515 of the fad2-F gene, which results in thesubstitution of a histidine residue for leucine in a KYLNNP (SEQ IDNO:27) motif (amino acid 172 of the Fad2 F polypeptide). Such a mutationis found within the variety Q508. See U.S. Pat. No. 6,342,658. Anotherexample of a fad2 mutation is the G to A mutation at nucleotide 908 ofthe fad2-F gene, which results in the substitution of a glutamic acidfor glycine in the DRDYGILNKV (SEQ ID NO:28) motif (amino acid 303 ofthe Fad2 F polypeptide). Such a mutation is found within the line Q4275,which has been deposited with the ATCC under Accession No. 97569. SeeU.S. Pat. No. 6,342,658. Another example of a suitable fad2 mutation canbe the C to T mutation at nucleotide 1001 of the fad2-F gene (asnumbered from the ATG), which results in the substitution of anisoleucine for threonine (amino acid 334 of the Fad2 F polypeptide).Such a mutation is found within the high oleic acid line Q7415.

Typically, the presence of one of the fad2-D or fad2-F mutations confersa mid-oleic acid phenotype (e.g., 70-80% oleic acid) to the seed oil,while the presence of both fad2-D and fad2-F mutations confers a higheroleic acid phenotype (e.g., >80% oleic acid). For example, Q4275contains the fad2-D mutation from IMC129 and a fad2-F mutation at aminoacid 303. Q508 contains fad2-D mutation from IMC129 and a fad2-Fmutation at amino acid 172. Q7415 contains the fad2-D mutation fromIMC129 and a fad2-F mutation at amino acid 334. The presence of bothfad2 mutations in Q4275, Q508, and Q7415 confers a high oleic acidphenotype of greater than 80% oleic acid.

Thus, in some embodiments, a Brassica plant contains a modified alleleat a fad3E locus and a modified allele at a fad2 locus. For example, aBrassica plant can contain a modified allele at a fad3E locus and amodified allele at a fad2 locus described above. A Brassica plant alsocan contain a modified allele at a fad3E locus, a modified allele at afad2 locus, and a modified allele at a fatA2 locus. A Brassica plant cancontain a modified allele at a fad3E locus, modified alleles at two ormore different fatB loci (three or four different loci), and a fad2locus described above. A Brassica plant also can contain a modifiedallele at a fad3E locus, fatA2 locus, modified alleles at two or moredifferent fatB loci (three or four different loci) and a modified alleleat a fad2 locus described above. In some embodiments, a Brassica plantcontains a modified allele at a fad3E locus, at least one modifiedallele at a different fad3 locus, a modified allele at a fatA2 locus, amodified allele at one or more different fatB loci (e.g., two or more),and a modified allele at one or more fad2 loci. A Brassica plant alsocan contain modified alleles at a fad3E locus and a fad3D locus,modified alleles at two or more different fatB loci (three or fourdifferent loci), modified alleles at fad2 loci, and modified alleles atfad3A and/or fad3B loci described above. A Brassica plant also contain amodified allele at a fad3E locus, a modified allele at a fad3D locus, amodified allele at a fatA2 locus, modified alleles at two or moredifferent FATB loci (three or four different loci), modified alleles atfad2 loci, and modified alleles at fad3A and fad3B loci described above.

One commercially important Brassica crop is B. napus. Commercial B.napus lines may be classified as either spring lines or winter lines.Winter lines are commonly planted in the autumn and flower in the springafter a period of vernalization over the winter. Spring lines do notrequire vernalization to flower and are commonly planted and harvestedin the same growing season. Winter lines are common in Europe, but mostwinter lines fare poorly in the colder winters of Canada and thenorthern United States. As a consequence, most B. napus growncommercially in North America are spring lines. One useful embodimentprovides a Brassica plant that is a B. napus plant. Though the B. napusplant may have a winter flowering habit, one preferred implementationhas a spring growing habit, i.e., it does not require vernalization toflower.

Production of Hybrid Brassica Varieties

Hybrid Brassica varieties can be produced by preventing self-pollinationof female parent plants (i.e., seed parents), permitting pollen frommale parent plants to fertilize such female parent plants, and allowingF₁ hybrid seeds to form on the female plants. Self-pollination of femaleplants can be prevented by emasculating the flowers at an early stage offlower development. Alternatively, pollen formation can be prevented onthe female parent plants using a form of male sterility. For example,male sterility can be cytoplasmic male sterility (CMS), nuclear malesterility, molecular male sterility wherein a transgene inhibitsmicrosporogenesis and/or pollen formation, or be produced byself-incompatibility. Female parent plants containing CMS areparticularly useful. CMS can be, for example of the ogu (Ogura), nap,pol, tour, or mur type. See, for example, Pellan-Delourme and Renard,1987, Proc. 7^(th) Int. Rapeseed Conf., Poznan, Poland, p. 199-203 andPellan-Delourme and Renard, 1988, Genome 30:234-238, for a descriptionof Ogura type CMS. See, Riungu and McVetty, 2003, Can. J. Plant Sci.,83:261-269 for a description of nap, pol, tour, and mur type CMS.

In embodiments in which the female parent plants are CMS, the maleparent plants typically contain a fertility restorer gene to ensure thatthe F₁ hybrids are fertile. For example, when the female parent containsan Ogura type CMS, a male parent is used that contains a fertilityrestorer gene that can overcome the Ogura type CMS. Non-limitingexamples of such fertility restorer genes include the Kosena typefertility restorer gene (U.S. Pat. No. 5,644,066) and Ogura fertilityrestorer genes (U.S. Pat. Nos. 6,229,072 and 6,392,127). In otherembodiments in which the female parents are CMS, male parents can beused that do not contain a fertility restorer. F₁ hybrids produced fromsuch parents are male sterile. Male sterile hybrid seed can beinter-planted with male fertile seed to provide pollen for seed-set onthe resulting male sterile plants.

The methods described herein can be used to form single-cross BrassicaF₁ hybrids. In such embodiments, the parent plants can be grown assubstantially homogeneous adjoining populations to facilitate naturalcross-pollination from the male parent plants to the female parentplants. The F₁ seed formed on the female parent plants is selectivelyharvested by conventional means. One also can grow the two parent plantsin bulk and harvest a blend of F₁ hybrid seed formed on the femaleparent and seed formed upon the male parent as the result ofself-pollination. Alternatively, three-way crosses can be carried outwherein a single-cross F₁ hybrid is used as a female parent and iscrossed with a different male parent that satisfies the fatty acidparameters for the female parent of the first cross. Here, assuming abulk planting, the overall oleic acid content of the vegetable oil maybe reduced over that of a single-cross hybrid; however, the seed yieldwill be further enhanced in view of the good agronomic performance ofboth parents when making the second cross. As another alternative,double-cross hybrids can be created wherein the F₁ progeny of twodifferent single-crosses are themselves crossed. Self-incompatibilitycan be used to particular advantage to prevent self-pollination offemale parents when forming a double-cross hybrid.

Hybrids described herein have good agronomic properties and exhibithybrid vigor, which results in seed yields that exceed that of eitherparent used in the formation of the F₁ hybrid. For example, yield can beat least 10% (e.g., 10% to 20%, 10% to 15%, 15% to 20%, or 25% to 35%)above that of either one or both parents. In some embodiments, the yieldexceeds that of open-pollinated spring canola varieties such as 46A65(Pioneer) or Q2 (University of Alberta), when grown under similargrowing conditions. For example, yield can be at least 10% (e.g., 10% to15% or 15% to 20%) above that of an open-pollinated variety.

Hybrids described herein typically produce seeds having very low levelsof glucosinolates (<30 μmol/gram of de-fatted meal at a moisture contentof 8.5%). In particular, hybrids can produce seeds having <20 μmol ofglucosinolates/gram of de-fatted meal. As such, hybrids can incorporatemutations that confer low glucosinolate levels. See, for example, U.S.Pat. No. 5,866,762. Glucosinolate levels can be determined in accordancewith known techniques, including high performance liquid chromatography(HPLC), as described in ISO 9167-1:1992(E), for quantification of total,intact glucosinolates, and gas-liquid chromatography for quantificationof trimethylsilyl (TMS) derivatives of extracted and purifieddesulfoglucosinolates. Both the HPLC and TMS methods for determiningglucosinolate levels analyze de-fatted or oil-free meal.

Canola Oil

Brassica plants disclosed herein are useful for producing canola oilswith low ALA content. For example, oil obtained from seeds of Brassicaplants described herein may have an ALA content of 0.5% to 1.6% (e.g.,0.5 to 1.5%, 0.5 to 1.0%, 0.5 to 0.8%, 0.6 to 1.4%, 0.6 to 1.3%, 0.6 to1.2%, 0.6 to 1.1%, 0.6 to 1.0%, 0.6 to 0.8%, 0.7 to 1.2%, 0.7 to 1.1%,0.8 to 1.2%, or 0.8 to 1.0%). In some embodiments, Brassica plantsdescribed herein produce canola oils with low ALA content (e.g., 0.5 to1.6%) and low or no total saturated fatty acids. For example, oilobtained from seeds of Brassica plants described herein may have an ALAcontent of 0.5 to 1.5% and a total saturated fatty acid content of 2.5to 6%, 3 to 5%, 3 to 4.5%, 3.25 to 3.75%, 3.0 to 3.5%, 3.4 to 3.7%, 3.6to 5%, 4 to 5.5%, 4 to 5%, or 4.25 to 5.25%. The palmitic acid contentof such oils can be 2.4 to 3.5% (e.g., 2.5 to 3% or 2.7 to 3.3%). Thestearic acid content of such oils can be 0.7 to 2.5% (e.g., 0.8 to 1.7%,0.9 to 1.5%, or 1.0 to 1.5%).

In some embodiments, an oil has an ALA content of 0.5 to 1.5%, an oleicacid content of 60 to 70% (e.g., 62 to 68%, 63 to 67%, or 65 to 66%),and a total saturated fatty acid content of 5 to 10%. In someembodiments, an oil has an ALA content of 0.6 to 1.5% (e.g., 0.7 to1.4%, 0.8 to 1.3%, or 0.9 to 1.2%) and an oleic acid content of 71 to80% (e.g., 72 to 78%, 72 to 76%, 73 to 75%, 74 to 77%, 74 to 78%, or 75to 80%). The total saturated content of such an oil can be 3 to 8%(e.g., 4 to 6%, 4 to 5.5%, 4 to 5%, 5 to 7%, 6 to 8%, or 7 to 8%). Insome embodiments, a canola oil can have an ALA content of 0.5 to 1.5%,an oleic acid content of 85 to 87% (e.g., 86 to 87%), and a totalsaturated fatty acid content of 5 to 6%. In some embodiments, an oil hasan ALA content of 0.5 to 1.5%, an oleic acid content of 81 to 90% (e.g.,82 to 88% or 83 to 87%) oleic acid and a total saturated fatty acidcontent of 3.5 to 4.5% (e.g., 3.75 to 4.25%, 3.9 to 4.1%, or 4.0%).

Oils described herein can have an eicosenoic acid content of 1.0 to1.9%. For example, an oil can have an eicosenoic acid content of 1.0 to1.4%, 1.1 to 1.3%, 1.1 to 1.6%, 1.2 to 1.6%, 1.4 to 1.9%, in addition toa low ALA content.

Oils described herein can have a linoleic acid content of 3.5 to 26%,e.g., 3.7 to 4.5%, 8 to 10%, 9 to 12%, 10 to 13%, 11 to 13%, 12 to 16%,13 to 16%, 14 to 18%, or 14 to 22%, in addition to a low ALA content.

Oils described herein have an erucic acid content of less than 2% (e.g.,less than 1%, 0.5%, 0.2, or 0.1%) in addition to a low ALA content.

The fatty acid composition of seeds can be determined by first crushingand extracting oil from seed samples (e.g., bulk seeds samples of 10 ormore seeds). TAGs in the seed are hydrolyzed to produce free fattyacids, which then can be converted to fatty acid methyl esters andanalyzed using techniques known to the skilled artisan, e.g., gas-liquidchromatography (GLC) according to AOCS Procedure Ce 1e-91. Near infrared(NIR) analysis can be performed on whole seed according to AOCSProcedure Am-192 (revised 1999)

Seeds harvested from plants described herein can be used to make a crudecanola oil or a refined, bleached, and deodorized (RBD) canola oil witha low ALA content. Harvested canola seed can be crushed to extract crudeoil and, if desired, refined, bleached and deodorized by techniquesknown in the art. See, e.g., Bailey's Industrial Oil and Fat Products,Volume 5, “Edible Fat and Oil Products: Processing Technologies” (6thEdition, 2005). Briefly, refining refers to removing most if not allfree fatty acids and other impurities such as phosphatides or proteinsubstances from a crude oil. One common method of refining involvestreating an oil with a strong base, followed by extensive washings withwater. Bleaching refers to a process that removes natural pigments(e.g., carotenoids, chlorophylls, and xanthophylls) and other impuritiessuch as metal cations (e.g., Fe, Cu and Zn). Bleaching can be done byabsorbing such pigments and/or cations on a natural bleaching earth orclay, which is usually added to an oil under vacuum and hightemperature. Deodorizing refers to the removal of relatively volatiletrace components (e.g., ketones, aldehydes, alcohols) from an oil thatcontribute to flavor, odor, and color. Deodorizing is usually done byinjecting steam into an oil heated to high temperatures (e.g., about470° F. to about 510° F.) under high vacuum (e.g., <5 mm Hg).

In one useful example, the seed can be tempered by spraying the seedwith water to raise the moisture to, for example, 8.5%. The temperedseed can be flaked using a smooth roller with, for example, a gapsetting of 0.23 to 0.27 mm. Heat may be applied to the flakes todeactivate enzymes, facilitate further cell rupturing, coalesce the oildroplets, or agglomerate protein particles in order to ease theextraction process. Typically, oil is removed from the heated canolaflakes by a screw press to press out a major fraction of the oil fromthe flakes. The resulting press cake contains some residual oil.

Crude oil produced from the pressing operation typically is passedthrough a settling tank with a slotted wire drainage top to remove thesolids expressed out with the oil in the screw pressing operation. Theclarified oil can be passed through a plate and frame filter to removethe remaining fine solid particles. Further oil can be extracted fromthe press cake produced from the screw pressing operation using knownsolvent extraction techniques, e.g., using commercial n-hexaneextraction. The canola oil recovered from the solvent extraction processis combined with the clarified oil from the screw pressing operation,resulting in a blended crude oil.

Free fatty acids and gums typically are removed from the crude oil byheating in a batch refining tank to which food grade phosphoric acid hasbeen added. The acid serves to convert the non-hydratable phosphatidesto a hydratable form, and to chelate minor metals that are present inthe crude oil. The phosphatides and the metal salts are removed from theoil along with the soapstock. The oil-acid mixture is treated withsodium hydroxide solution to neutralize the free fatty acids and thephosphoric acid in the acid-oil mixture. The neutralized free fattyacids, phosphatides, etc. (soapstock) are drained off from theneutralized oil. A water wash may be done to further reduce the soapcontent of the oil. The oil may be bleached and deodorized before use,if desired, by techniques known in the art.

Oils obtained from plants described herein can have increased oxidativestability, which can be measured using, for example, an OxidativeStability Index Instrument (e.g., from Omnion, Inc., Rockland, Mass.)according to AOCS Official Method Cd 12b-92 (revised 1993). Oxidativestability is often expressed in terms of “AOM” hours.

Oils obtained from plants described herein also can have increasedflavor stability, which can be measured using, for example, trained testpanels in room-odor tests according to Mounts, J. Am. Oil Chem. Soc.56:659-663, 1979 and the AOCS Recommended Practice Cg 2-83 for theFlavor Evaluation of Vegetable Oils (Methods and Standard Practices ofthe AOCS, 4th Edition (1989)). The technique encompasses standard samplepreparation and presentation, as well as reference standards and methodfor scoring oils.

Food Compositions

This document also features food compositions containing the oilsdescribed above. For example, oils having a low ALA content (e.g., 0.5to 1.5%) can be used for food applications or for frying. Oils having alow ALA content in combination with a low (6% or less) or very low (3.5%or less) total saturated fatty acid content can be used to replace orreduce the amount of saturated fatty acids and hydrogenated oils (e.g.,partially hydrogenated oils) in various food products such that thelevels of saturated fatty acids and trans fatty acids are reduced in thefood products. In particular, canola oils having a low ALA content incombination with a low total saturated fatty acid content and a mid orhigh oleic acid content can be used to replace or reduce the amount ofsaturated fats and partially hydrogenated oils in processed or packagedfood products, including bakery products such as cookies, muffins,doughnuts, pastries (e.g., toaster pastries), pie fillings, pie crusts,pizza crusts, frostings, breads, biscuits, and cakes, breakfast cereals,breakfast bars, puddings, and crackers.

For example, an oil described herein can be used to produce sandwichcookies that contain no or reduced levels of partially hydrogenated oilsin the cookie and/or crème filling. In some embodiments, the cookiesalso have a reduced total saturated fatty acid content. Such cookiecompositions can include, for example, in addition to canola oil, flour,sweetener (e.g., sugar, molasses, honey, high fructose corn syrup,artificial sweetener such as sucralose, saccharine, aspartame, oracesulfame potassium, and combinations thereof), eggs, salt, flavorants(e.g., chocolate, vanilla, or lemon), a leavening agent (e.g., sodiumbicarbonate or other baking acid such as monocalcium phosphatemonohydrate, sodium aluminum sulfate, sodium acid pyrophosphate, sodiumaluminum phosphate, dicalcium phosphate, glucano-deltalactone, orpotassium hydrogen tartrate, or combinations thereof), and optionally,an emulsifier (e.g., mono- and diglycerides of fatty acids, propyleneglycol mono- and di-esters of fatty acids, glycerol-lactose esters offatty acids, ethoxylated or succinylated mono- and diglycerides,lecithin, diacetyl tartaric acid esters or mono- and diglycerides,sucrose esters of glycerol, and combinations thereof). A crème fillingcomposition can include, in addition to canola oil, sweetener (e.g.,powdered sugar, granulated sugar, honey, high fructose corn syrup,artificial sweetener, or combinations thereof), flavorant (e.g.,vanilla, chocolate, or lemon), salt, and, optionally, emulsifier.

Canola oils (e.g., with a low ALA, low total saturated fatty acid andlow or high oleic acid content) also are useful for frying applicationsdue to the polyunsaturated content, which is low enough to have improvedoxidative stability for frying yet high enough to impart the desiredfried flavor to the food being fried. For example, canola oils can beused to produce fried foods such as snack chips (e.g., corn or potatochips), French fries, or other quick serve foods.

Oils described herein also can be used to formulate spray coatings forfood products (e.g., cereals or snacks such as crackers). In someembodiments, the spray coating can include other vegetable oils such assunflower, cottonseed, corn, or soybean oils. A spray coating also caninclude an antioxidant and/or a seasoning.

Oils described herein also can be use in the manufacturing of dressings,mayonnaises, and sauces to provide a reduction in the total saturatedfat content of the product. Oils described herein can be used as a baseoil for creating structured fat solutions such as microwave popcornsolid fats or canola butter formulations.

Plant Breeding

The nucleic acids described herein (e.g., fad3D and fad3E nucleic acids)can be used as markers in plant genetic mapping and plant breedingprograms. Such markers may include restriction fragment lengthpolymorphism (RFLP), random amplified polymorphic DNA detection (RAPD),amplified fragment length polymorphism (AFLP), simple sequence repeat(SSR) or microsatellite, for example. Marker-assisted breedingtechniques may be used to identify and follow a desired fatty acidcomposition (e.g., low linolenic acid) during the breeding process. Forexample, a nucleic acid described herein, such as the nucleic acidsequence set forth in SEQ ID NO:1 or SEQ ID NO:32, or the complementthereof, can be used to identify one or more individual plants thatpossess the polymorphic allele correlated with the desired linolenicacid content. Those plants then can be used in a breeding program tocombine the polymorphic allele with a plurality of other alleles atother loci that are correlated with a desired variation (e.g., in fattyacid composition). In some embodiments, a fragment of the nucleic acidsequence set forth in SEQ ID NO:1 or SEQ ID NO:32, or the complementthereof, that is at least 50 nucleotides in length can be used todistinguish a modified fad3 allele from a wild-type Fad3 allele (e.g.,by allele-specific hybridization or by PCR).

Techniques suitable for use in a plant breeding program are known in theart and include, without limitation, backcrossing, mass selection,pedigree breeding, bulk selection, crossing to another population andrecurrent selection. These techniques can be used alone or incombination with one or more other techniques in a breeding program.Thus, each identified plant is selfed or crossed to a different plant toproduce seed, which is then germinated to form progeny plants. At leastone such progeny plant is then selfed or crossed with a different plantto form a subsequent progeny generation. The breeding program can repeatthe steps of selfing or outcrossing for an additional 0 to 5 generationsas appropriate in order to achieve the desired uniformity and stabilityin the resulting plant line, which retains the polymorphic allele. Inmost breeding programs, analysis for the particular polymorphic allelewill be carried out in each generation, although analysis can be carriedout in alternate generations if desired.

In some cases, selection for other useful traits is also carried out,e.g., selection for disease resistance. Selection for such other traitscan be carried out before, during or after identification of individualplants that possess the desired polymorphic allele.

Marker-assisted breeding techniques may be used in addition to, or as analternative to, other sorts of identification techniques. An example ofmarker-assisted breeding is the use of PCR primers that specificallyamplify a sequence containing a desired mutation in the fad3D or fad3Esequence.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES

In the Tables described herein, the fatty acids are referred to by thelength of the carbon chain and number of double bonds within the chain.For example, C14:0 refers to myristic acid; C16:0 refers to palmiticacid; C18:0 refers to stearic acid; C18:1 refers to oleic acid; C18:2refers to linoleic acid; C18:3 refers to ALA; C20:0 refers to archidicacid; C20:1 refers to eicosenoic acid; C22:0 refers to behenic acid;C22:1 refers to erucic acid; C24:0 refers to lignoceric acid; and C24:1refers to nervonic acid. “Total Sats” refers to the total of C14:0,C16:0, C18:0, C20:0, C22:0, and C24:0. Representative fatty acidprofiles are provided for each of the specified samples.

Unless otherwise indicated, all percentages refer to wt % based on totalwt % of fatty acids (i.e., fatty acid moieties) in the oil as determinedby measuring the FAME moieties in accordance with the modified versionof ROCS Ce 1c-89 set forth in Example 1.

Example 1 Brassica Plant Lines 1904 and 2558

Plants producing an oil with a low ALA content were obtained bysubjecting a population of B. napus IMC201 seeds to chemical mutagenesisand selecting for low linolenic acid content (<1.5%). The typical fattyacid composition of field grown IMC201 is 3.6% C16:0, 1.8% C18:0, 76%C18:1, 12.5% C18:2, 3% C18:3, 0.7% C20:0, 1.5% C20:1, 0.3% C22:0, 0%C22:1, with total saturates of 6.4%. Prior to mutagenesis, IMC201 seedswere pre-imbibed in 700 gm seed lots by soaking for 15 min then drainingfor 5 min at room temperature. This was repeated four times to softenthe seed coat. The pre-imbibed seed then were treated with 4 mM methylN-nitrosoguanidine (MNNG) for three hours. Following the treatment withMNNG, seeds were drained of the mutagen and rinsed with water for onehour. After removing the water, the seeds were treated with 52.5 mMethyl methanesulfonate (EMS) for sixteen hours. Following the treatmentwith EMS, the seeds were drained of mutagen and rinsed with water forone and one half hours. This dual mutagen treatment produced an LD₅₀with the seed population.

Lines 1904 and 2558 were selected from the mutagenized population ofIMC201 seeds as follows. Three thousand bulk M2 generation seeds wereplanted. Upon maturity, M3 seed (2500 individuals) was harvested from2500 M2 plants and analyzed via a modified method for gas chromatographdetermination of fatty acid profile per the American Oil Chemist'sSociety protocol (AOCS Ce 1c-89). In accordance with AOCS Ce 1c-89, theoil from the seeds was first treated to convert the acylglycerols tofatty acid methyl esters (“FAMEs”) and vials of the FAMEs were placed ina gas chromatograph for analysis in accordance with a modified versionof American Oil Chemist's Society Official Method Ce 1-62 that employedan Agilent 6890 gas chromatograph (Agilent Technologies, Santa Clara,Calif.) equipped with a fused silica capillary column (5 m×0.180 mm and0.20 μm film thickness) packed with a polyethylene glycol based DB-Wax®for liquid phase separation (J&W Scientific, Folsom, Calif.). Hydrogen(H2) was used as the carrier gas at a flow rate of 2.5 mL/min and thecolumn temperature was isothermal at 200° C. Seed from each plant wastested via this method in replicates of two.

Lines 1904 and 2558 were identified as having low linolenic acid contentin seed oil. M3 seeds of lines 1904 and 2558 were planted (50 per line)and the resulting plants were self pollinated. M4 seeds were harvestedfrom the plants and bulk seed samples (approximately 20 seeds) wereanalyzed via GC. The results are presented in Table I Lines 1904 and2558 had ALA contents ranging from approximately 0.70% to 1.95%. Line1904 was deposited with the American Type Culture Collection (ATCC)under Accession No. PTA-11273 and line 2558 was deposited with theAmerican Type Culture Collection (ATCC) under Accession No. PTA-11274.

TABLE 1 Fatty acid profile of harvested M4 generation mutant seed.RESCHID C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 C20:0 M3B-1904-010.065 4.675 0.323 2.307 72.509 16.181 0.912 0.818 M3B-1904-02 0.0524.183 0.256 2.507 74.995 13.520 1.431 0.911 M3B-1904-03 0.055 3.9380.216 2.272 75.548 13.762 1.239 0.834 M3B-1904-04 0.058 4.089 0.2182.313 74.369 14.354 1.507 0.862 M3B-1904-05 0.056 3.930 0.202 2.57175.823 13.172 1.377 0.889 M3B-1904-06 0.062 4.286 0.256 2.170 74.70014.110 1.195 0.829 M3B-1904-07 0.053 4.147 0.247 2.386 75.233 13.3051.306 0.866 M3B-1904-08 0.058 4.198 0.237 2.530 74.591 13.613 1.2170.965 M3B-1904-09 0.063 4.133 0.210 2.246 75.223 13.916 1.147 0.845M3B-1904-10 0.068 4.367 0.300 2.708 71.414 16.504 1.386 0.891M3B-1904-11 0.057 4.022 0.246 2.343 75.222 13.434 1.450 0.835M3B-1904-12 0.058 4.157 0.247 2.315 74.670 14.226 1.160 0.816M3B-1904-13 0.055 4.179 0.251 2.473 73.852 15.023 0.839 0.853M3B-1904-14 0.059 4.151 0.251 2.268 75.153 13.876 1.107 0.819M3B-1904-15 0.073 4.095 0.272 2.390 75.613 13.511 1.214 0.829M3B-1904-16 0.054 3.947 0.210 2.653 75.732 13.173 1.516 0.869M3B-1904-17 0.051 3.877 0.236 2.634 75.262 13.495 0.809 0.942M3B-1904-18 0.057 3.901 0.240 2.598 75.561 13.259 1.268 0.908M3B-1904-19 0.058 3.942 0.215 2.270 76.923 12.821 0.779 0.786M3B-1904-20 0.066 4.044 0.263 2.105 74.938 14.222 1.436 0.758M3B-1904-21 0.071 4.264 0.275 2.275 74.864 14.105 1.123 0.852M3B-1904-22 0.060 4.242 0.257 2.308 75.173 13.872 1.137 0.817M3B-1904-23 0.060 4.095 0.240 2.122 73.657 15.742 0.780 0.814M3B-1904-24 0.068 4.046 0.272 2.294 74.481 14.038 1.251 0.848M3B-1904-25 0.063 4.162 0.265 2.364 75.169 14.022 1.126 0.825M3B-1904-26 0.054 3.981 0.246 2.228 76.741 13.190 0.741 0.795M3B-1904-27 0.057 4.058 0.240 2.112 75.577 14.077 0.844 0.824M3B-1904-28 0.058 4.221 0.274 2.259 74.558 13.776 1.219 0.837M3B-1904-29 0.058 4.364 0.223 2.550 74.320 14.035 1.298 0.941M3B-1904-30 0.054 4.133 0.225 2.419 74.744 13.928 1.317 0.926M3B-1904-31 0.070 4.209 0.259 2.225 76.060 13.424 1.118 0.755M3B-1904-32 0.079 4.867 0.385 3.113 69.744 16.279 1.951 1.048M3B-1904-33 0.066 4.346 0.261 2.741 74.104 13.778 1.582 0.882M3B-1904-34 0.079 4.571 0.269 2.605 73.843 14.412 1.272 0.889M3B-1904-35 0.067 4.168 0.259 2.460 75.821 13.653 0.744 0.808M3B-1904-36 0.051 4.128 0.209 2.338 76.117 12.715 1.416 0.894M3B-1904-37 0.054 4.238 0.201 2.295 74.756 13.803 1.531 0.891M3B-1904-38 0.057 4.325 0.237 2.472 75.481 13.396 1.460 0.825M3B-1904-39 0.059 4.178 0.249 2.392 74.176 14.528 1.360 0.890M3B-1904-40 0.056 4.176 0.245 3.409 72.309 13.105 1.378 1.171M3B-1904-41 0.057 4.141 0.239 2.392 75.487 13.894 1.077 0.841M3B-1904-42 0.054 3.947 0.225 2.488 74.792 14.490 1.131 0.831M3B-1904-43 0.051 3.985 0.226 2.263 75.686 13.277 1.570 0.823M3B-1904-44 0.052 4.137 0.202 2.677 75.733 12.649 1.506 0.950M3B-1904-45 0.052 3.929 0.195 2.280 75.101 14.334 1.117 0.836M3B-1904-46 0.060 4.354 0.283 2.577 73.385 13.857 1.635 0.975M3B-1904-47 0.062 4.373 0.291 2.473 74.242 13.602 1.288 0.870M3B-1904-48 0.054 4.074 0.233 2.174 75.247 13.779 1.143 0.830M3B-1904-49 0.059 4.125 0.253 2.093 74.920 14.379 0.909 0.804M3B-1904-50 0.060 4.157 0.235 2.037 74.522 14.402 1.539 0.786M3B-2558-01 0.067 3.772 0.244 3.251 78.153 10.081 0.785 1.208M3B-2558-02 0.058 3.674 0.218 2.989 78.233 10.027 1.181 1.095M3B-2558-03 0.062 4.123 0.308 3.329 76.058 11.181 1.293 1.209M3B-2558-04 0.065 4.134 0.295 2.915 75.964 12.077 0.974 1.029M3B-2558-05 0.058 4.108 0.283 3.267 77.220 10.413 1.176 1.160M3B-2558-06 0.064 3.801 0.254 3.057 78.311 9.731 1.076 1.094 M3B-2558-070.048 3.660 0.231 2.788 76.319 12.244 1.262 1.023 M3B-2558-08 0.0693.845 0.275 3.494 76.433 10.690 1.309 1.271 M3B-2558-09 0.055 3.8520.281 2.987 77.339 10.931 1.184 0.988 M3B-2558-10 0.055 3.864 0.2673.079 77.765 9.847 1.509 1.103 M3B-2558-11 0.056 3.833 0.261 3.04378.849 9.032 0.933 1.164 M3B-2558-12 0.049 3.996 0.218 2.955 77.21811.409 0.814 1.056 M3B-2558-13 0.057 3.899 0.246 3.409 76.902 10.9341.205 1.135 M3B-2558-14 0.056 3.984 0.223 3.775 78.095 8.899 1.184 1.323M3B-2558-15 0.052 3.762 0.231 3.088 77.908 10.410 1.186 1.078M3B-2558-16 0.053 3.865 0.245 3.360 77.868 9.754 1.223 1.176 M3B-2558-170.056 4.070 0.244 3.064 78.090 10.426 1.190 1.061 M3B-2558-18 0.0734.064 0.259 2.869 76.821 11.402 0.878 1.053 M3B-2558-19 0.062 3.8350.250 3.165 76.790 11.196 1.159 1.143 M3B-2558-20 0.071 3.987 0.2893.482 76.133 10.992 1.238 1.240 M3B-2558-21 0.072 4.100 0.341 3.14776.942 10.636 1.212 1.111 M3B-2558-22 0.055 3.875 0.279 2.700 76.78011.604 1.486 0.930 M3B-2558-23 0.063 4.113 0.266 3.179 76.201 11.5270.962 1.128 M3B-2558-24 0.058 3.779 0.252 2.895 77.381 10.867 1.1611.035 M3B-2558-25 0.054 4.008 0.266 3.123 76.541 11.413 1.142 1.115M3B-2558-26 0.063 3.956 0.283 3.197 76.084 11.172 1.262 1.166M3B-2558-27 0.057 4.003 0.297 2.820 74.978 13.117 1.192 0.989M3B-2558-28 0.061 3.837 0.277 3.584 75.859 11.272 1.217 1.235M3B-2558-29 0.056 3.879 0.275 2.685 75.620 12.874 1.307 0.977M3B-2558-31 0.059 3.933 0.266 2.919 76.965 11.336 0.918 1.097M3B-2558-32 0.059 3.876 0.287 3.005 76.378 11.972 0.854 1.017M3B-2558-33 0.060 4.204 0.271 3.251 74.489 12.869 1.378 1.132M3B-2558-34 0.089 4.307 0.314 3.290 73.974 13.250 1.204 1.106M3B-2558-35 0.053 3.747 0.208 3.144 77.721 10.975 0.722 1.128M3B-2558-36 0.056 4.071 0.281 2.911 76.432 11.760 1.244 1.027M3B-2558-37 0.053 3.931 0.265 3.287 76.237 11.366 1.446 1.141M3B-2558-39 0.067 3.998 0.303 3.160 75.020 12.561 1.320 1.066M3B-2558-40 0.051 3.895 0.251 3.493 76.324 10.978 1.476 1.190M3B-2558-41 0.068 3.709 0.257 3.452 76.017 11.107 1.491 1.183M3B-2558-42 0.062 3.932 0.258 3.214 76.168 11.541 1.138 1.094M3B-2558-43 0.054 3.846 0.248 3.215 75.801 12.266 1.135 1.090M3B-2558-44 0.051 3.782 0.279 3.271 76.059 11.827 1.487 1.082M3B-2558-45 0.060 3.787 0.252 3.053 76.253 11.901 1.163 1.007M3B-2558-46 0.054 3.758 0.255 3.377 78.569 9.676 0.844 1.183 M3B-2558-470.060 3.981 0.277 3.142 76.833 11.297 0.823 1.082 M3B-2558-48 0.0523.816 0.264 3.204 76.778 11.235 1.305 1.112 M3B-2558-49 0.053 4.0680.277 3.172 75.712 11.904 1.645 1.054 M3B-2558-50 0.059 4.063 0.2823.440 75.763 11.199 1.607 1.126 TOT RESCHID C20:1 C20:2 C22:0 C22:1C24:0 C24:1 SATS M3B-1904-01 1.068 0.046 0.410 0.000 0.210 0.475 8.486M3B-1904-02 1.180 0.046 0.453 0.000 0.267 0.201 8.372 M3B-1904-03 1.2840.048 0.421 0.000 0.250 0.133 7.771 M3B-1904-04 1.234 0.052 0.437 0.0170.243 0.247 8.003 M3B-1904-05 1.133 0.044 0.407 0.000 0.210 0.186 8.063M3B-1904-06 1.206 0.050 0.463 0.000 0.258 0.415 8.068 M3B-1904-07 1.2590.047 0.443 0.000 0.219 0.491 8.113 M3B-1904-08 1.315 0.052 0.526 0.0310.307 0.359 8.584 M3B-1904-09 1.215 0.048 0.436 0.021 0.237 0.260 7.959M3B-1904-10 1.190 0.060 0.443 0.000 0.304 0.364 8.780 M3B-1904-11 1.2280.045 0.418 0.000 0.220 0.481 7.895 M3B-1904-12 1.215 0.045 0.395 0.0000.198 0.498 7.939 M3B-1904-13 1.227 0.051 0.418 0.000 0.238 0.540 8.217M3B-1904-14 1.190 0.049 0.433 0.000 0.249 0.394 7.979 M3B-1904-15 1.1440.042 0.389 0.000 0.207 0.222 7.983 M3B-1904-16 1.146 0.043 0.374 0.0000.181 0.104 8.077 M3B-1904-17 1.277 0.051 0.480 0.000 0.266 0.621 8.250M3B-1904-18 1.190 0.046 0.434 0.000 0.228 0.311 8.126 M3B-1904-19 1.1530.046 0.389 0.000 0.198 0.423 7.642 M3B-1904-20 1.215 0.044 0.373 0.0000.217 0.320 7.561 M3B-1904-21 1.193 0.044 0.424 0.000 0.245 0.265 8.131M3B-1904-22 1.130 0.045 0.392 0.000 0.208 0.361 8.025 M3B-1904-23 1.3600.059 0.451 0.000 0.284 0.336 7.825 M3B-1904-24 1.372 0.048 0.468 0.0360.238 0.541 7.962 M3B-1904-25 1.134 0.042 0.384 0.000 0.208 0.235 8.007M3B-1904-26 1.133 0.042 0.398 0.000 0.193 0.259 7.648 M3B-1904-27 1.3110.050 0.466 0.024 0.233 0.127 7.750 M3B-1904-28 1.348 0.049 0.458 0.0000.230 0.713 8.063 M3B-1904-29 1.236 0.051 0.484 0.000 0.289 0.150 8.687M3B-1904-30 1.296 0.054 0.501 0.000 0.264 0.140 8.297 M3B-1904-31 1.0100.043 0.348 0.000 0.165 0.316 7.771 M3B-1904-32 1.272 0.066 0.548 0.0000.350 0.300 10.005 M3B-1904-33 1.079 0.049 0.405 0.000 0.253 0.455 8.693M3B-1904-34 1.063 0.050 0.430 0.000 0.197 0.319 8.771 M3B-1904-35 1.0690.040 0.356 0.000 0.171 0.386 8.029 M3B-1904-36 1.240 0.048 0.467 0.0000.245 0.132 8.122 M3B-1904-37 1.301 0.049 0.471 0.000 0.266 0.145 8.215M3B-1904-38 1.062 0.045 0.357 0.000 0.181 0.103 8.216 M3B-1904-39 1.2570.047 0.456 0.000 0.288 0.121 8.262 M3B-1904-40 1.309 0.054 0.610 0.0000.400 1.777 9.823 M3B-1904-41 1.121 0.044 0.410 0.000 0.195 0.103 8.036M3B-1904-42 1.242 0.046 0.388 0.000 0.220 0.145 7.928 M3B-1904-43 1.2980.046 0.425 0.000 0.203 0.146 7.751 M3B-1904-44 1.217 0.046 0.456 0.0000.221 0.155 8.493 M3B-1904-45 1.307 0.047 0.445 0.000 0.225 0.133 7.767M3B-1904-46 1.233 0.053 0.550 0.000 0.317 0.721 8.833 M3B-1904-47 1.2320.047 0.451 0.000 0.246 0.824 8.475 M3B-1904-48 1.333 0.051 0.454 0.0200.266 0.343 7.851 M3B-1904-49 1.281 0.057 0.457 0.000 0.248 0.416 7.786M3B-1904-50 1.209 0.053 0.425 0.015 0.233 0.329 7.698 M3B-2558-01 1.3130.037 0.583 0.016 0.354 0.137 9.234 M3B-2558-02 1.326 0.045 0.547 0.0160.305 0.288 8.668 M3B-2558-03 1.278 0.045 0.604 0.000 0.362 0.150 9.689M3B-2558-04 1.325 0.046 0.509 0.000 0.304 0.364 8.956 M3B-2558-05 1.2590.044 0.545 0.000 0.332 0.136 9.469 M3B-2558-06 1.302 0.039 0.523 0.0000.292 0.456 8.831 M3B-2558-07 1.394 0.059 0.511 0.000 0.254 0.208 8.283M3B-2558-08 1.372 0.044 0.643 0.000 0.374 0.181 9.696 M3B-2558-09 1.2060.038 0.442 0.000 0.232 0.464 8.556 M3B-2558-10 1.261 0.041 0.539 0.0000.303 0.369 8.942 M3B-2558-11 1.377 0.040 0.618 0.000 0.388 0.408 9.102M3B-2558-12 1.282 0.047 0.516 0.000 0.300 0.140 8.872 M3B-2558-13 1.2540.044 0.523 0.000 0.297 0.094 9.321 M3B-2558-14 1.297 0.040 0.637 0.0000.350 0.137 10.125 M3B-2558-15 1.305 0.045 0.521 0.000 0.297 0.117 8.797M3B-2558-16 1.231 0.038 0.545 0.000 0.329 0.313 9.328 M3B-2558-17 1.2840.000 0.516 0.000 0.000 0.000 8.767 M3B-2558-18 1.301 0.041 0.530 0.0000.276 0.433 8.866 M3B-2558-19 1.325 0.043 0.546 0.000 0.314 0.172 9.065M3B-2558-20 1.328 0.042 0.593 0.000 0.384 0.220 9.758 M3B-2558-21 1.2470.040 0.537 0.000 0.340 0.276 9.306 M3B-2558-22 1.214 0.039 0.427 0.0000.215 0.396 8.201 M3B-2558-23 1.313 0.045 0.576 0.000 0.340 0.287 9.399M3B-2558-24 1.302 0.045 0.514 0.023 0.307 0.381 8.588 M3B-2558-25 1.3020.046 0.535 0.017 0.304 0.134 9.140 M3B-2558-26 1.310 0.047 0.607 0.0170.363 0.474 9.351 M3B-2558-27 1.321 0.049 0.506 0.027 0.288 0.356 8.663M3B-2558-28 1.294 0.044 0.596 0.025 0.353 0.348 9.666 M3B-2558-29 1.3270.056 0.510 0.000 0.288 0.147 8.394 M3B-2558-31 1.354 0.048 0.583 0.0000.367 0.155 8.958 M3B-2558-32 1.250 0.042 0.490 0.000 0.273 0.499 8.719M3B-2558-33 1.289 0.054 0.564 0.000 0.301 0.138 9.513 M3B-2558-34 1.2390.047 0.518 0.000 0.284 0.379 9.593 M3B-2558-35 1.312 0.042 0.532 0.0000.296 0.121 8.899 M3B-2558-36 1.287 0.050 0.523 0.000 0.243 0.115 8.831M3B-2558-37 1.281 0.045 0.519 0.000 0.306 0.124 9.237 M3B-2558-39 1.2830.048 0.515 0.000 0.303 0.356 9.110 M3B-2558-40 1.281 0.046 0.557 0.0000.335 0.125 9.520 M3B-2558-41 1.251 0.044 0.577 0.000 0.344 0.502 9.332M3B-2558-42 1.247 0.044 0.525 0.000 0.284 0.493 9.111 M3B-2558-43 1.3390.049 0.521 0.000 0.309 0.128 9.034 M3B-2558-44 1.226 0.049 0.492 0.0000.277 0.120 8.954 M3B-2558-45 1.259 0.047 0.473 0.015 0.259 0.472 8.638M3B-2558-46 1.269 0.036 0.551 0.000 0.318 0.110 9.241 M3B-2558-47 1.2610.038 0.510 0.000 0.291 0.405 9.065 M3B-2558-48 1.282 0.045 0.531 0.0000.265 0.111 8.980 M3B-2558-49 1.188 0.047 0.493 0.000 0.273 0.114 9.114M3B-2558-50 1.215 0.042 0.503 0.000 0.272 0.431 9.462

Selected M4 individuals were self pollinated to generate M5 seeds andfurther evaluated in an environmentally controlled plant growth chamber.Seeds from M3B-2558-35 and M3-B1904-35 were planted in Premier Pro-MixBX potting soil (Premier Horticulture, Quebec, Canada) in four inchplastic pots. Planted seeds were watered and stratified at 5° C. for 5days and germinated at 20° C. day temperature and 17° C. nighttemperature (20/17) in Conviron ATC60 controlled-environment growthchambers (Controlled Environments Limited, Winnipeg, MB). Each genotypecombination was randomized and replicated 10 times in each of twoseparate growth chambers. At flowering, one chamber was reduced to adiurnal temperature cycle of 15° C. day temperature and 12° C. nighttemperature (15/12) while the other remained at 20/17. The temperaturetreatments were imposed to identify the effects of temperature on fattyacid composition. Plants were watered five times per week and fertilizedbi-weekly using a 20:20:20 (NPK) liquid fertilizer at a rate of 150 ppm.Plants were bagged individually to ensure self pollination and geneticpurity of the seed. Seeds from each plant were harvested individually atphysiological seed maturity. The fatty acid profile of the seeds wasdetermined using the modified GC method described above (replicates oftwo).

Fatty acid data from plants grown under the different temperatureregimes was analyzed in two ways. First, data was analyzed separately asdifferent environments and then it was pooled and analyzed acrossenvironments. Data was analyzed in SAS (SAS Institute, 2003) using procglm to estimate differences in mean fatty acid values. Table 2 containsthe population size, mean value and standard deviation of oleic,linoleic and linolenic fatty acid of seeds produced by plants carryingmutant fad3 alleles and grown in two environmental growth chambers setat different diurnal temperature regimes (20° C. day/17° C. night; 15°C. day/12° C. night) as discussed above. Genotypes 1904-35 and 2558-35are mutant allele combinations and v1030 hybrid and IMC02 are controls.The 1904-35, 2558-35, and IMC02 lines each contain mutant fad3A andfad3B alleles, while line 1904-35 also contains a mutant fad3E alleleand a mutant fad3D allele (see below). Means with different letters aresignificantly different as determined by a Student-Newman-Keuls meanseparation test. In conclusion, lines 1904-35 and 2558-35 can reach analpha-linolenic content less than v1030 and IMC02.

Seeds of lines 1904 and 2558 were deposited with the American TypeCulture Collection (ATCC) (Manassas, Va.) on Sep. 1, 2010, underconditions of the Budapest Treaty and assigned Accession Nos. PTA-11273and PTA-11274, respectively. All restrictions upon public access to thedeposits will be irrevocably removed upon grant of the patent. Thedeposits will be replaced if the depository cannot dispense viablesamples.

TABLE 2 Mean oleic, linoleic and linolenic acid content in twoenvironments Mean Mean Mean RESCHID C18:1 s.d. C18:2 s.d. C18:3 s.d. N15/12 Environment v1030 65.877 0.564 22.031 0.523 3.430 a 0.116 9 IMC0269.728 1.528 20.484 1.434 1.815 b 0.109 9 1904-35 73.986 1.437 16.9561.369 1.071 c 0.082 10 2558-35 77.276 1.191 13.051 1.505 0.976 d 0.08110 17/20 Environment v1030 65.053 1.397 22.906 1.570 2.952 a 0.133 10IMC02 72.211 1.604 17.543 1.986 1.378 b 0.098 10 1904-35 77.009 0.47513.477 0.489 1.052 c 0.040 9 2558-35 78.470 0.924 11.238 1.129 0.993 c0.080 10 Across Environments V1030 65.443 1.138 22.491 1.247 3.179 a0.274 19 IMC02 71.035 1.987 18.936 2.272 1.585 b 0.246 19 1904-35 75.4181.881 15.308 2.056 1.062 c 0.065 19 2558-35 77.873 1.205 12.145 1.5950.984 c 0.079 20

Example 2 Identification of a Fad3E Mutation in 1904-35 Plants

Genome mapping, map-based gene cloning, and direct-sequencing strategieswere used to identify loci associated with the <1.5% linolenic fattyacid content in the 1904-35 line described in Example 1. A DH (doubledhaploid) population was developed from a cross between 1904-35 and95CB504, a B line (maintainer). The two parental lines were screenedwith 1066 SNP (single nucleotide polymorphism) markers using theMassARRAY platform (Sequenom Inc., San Diego, Calif.) to identifypolymorphic SNP markers between the two parents; 174 polymorphic SNPmarkers were identified.

Single marker correlations and multiple regression analysis betweenfatty acid composition and SNP markers were carried out using the SASprogram (SAS Institute 1988). A Brassica napus genetic linkage map wasconstructed using the Kosambi function in JoinMap 3.0 (Kyazma). Intervalmapping for quantitative trait loci (QTL) was done with MapQTL 4.0(Kyazma). A LOD score >3.0 was considered as the significance thresholdto declare the association intervals.

Comparative genome mapping was performed to locate the identified QTL inBrassica napus chromosomes and further identify the Brassica rapa BAC(Bacterial Artificial Chromosome) clones encompassing the identified SNPmarkers and the candidate genes in the identified QTL interval for the<1.5% linolenic acid content using publicly available Brassica andArabidosis genome sequences, genes, genetic linkage maps, and otherinformation from the world wide web at brassica.bbsrc.ac.uk/, andncbi.nlm.nih.gov/.

A total of 217 DH lines were genotyped with 174 polymorphic SNP markers.QTL mapping identified two QTLs for low linolenic acid content (<1.5%C18:3). Comparative genome mapping located one QTL on the N3 chromosomein Brassica napus (A3 in Brassica rapa) and further identified a Fad3Ecandidate gene which is located at 1cM from the SNP marker that showedsignificant association with C18:3 content. The 1cM interval between theSNP marker and Fad3E gene is 248 kb according to co-linearity with theArabidopsis genome. Example 3 describes the second QTL on the N5chromosome in Brassica napus (A5 in Brassica rapa).

The Fad3E genes from chromosome N3 of the Brassica napus genome weresequenced from 1904-35, 95CB504 and IMC201. The sequences were analyzedusing BLAST (the Basic Local Alignment Search Tool) andDNASTAR/Lasergene 8.0 (DNASTAR, Inc). A single nucleotide substitutionwas identified in one of the two Fad3E isoforms from the 1904-35 mutantline that was not present in 95CB504 and IMC201. FIG. 1 shows thesequence alignment of the BnFad3E gene from 1904-35 and IMC201, and theBrFad3E located in Brassica rapa BAC, KBrH013B15 from the world wide webat brassica-rapa.org. The nucleotide substitution of a “A” in 1904-35for “G” in IMC201 and 95CB504 at position 1851 of this alignment(position 1756 in SEQ ID:NO:1). As shown in FIG. 2, this transitionmutation of Fad3E is at the exon 3, intron 3 border. FIG. 3 shows thealignment of FAD3E amino acid sequences from 1904 BnFAD3E-2 and IMC201BnFAD3E-2 (SEQ ID NO:29), BrFAD3E deduced from BrFad3E (world wide webat brassica-rapa.org) (SEQ ID NO:30), and AtFAD3 (GenBank accessionnumber: NP_(—)180559; SEQ ID NO:31). The fad3E-2 SNP allele results inan altered consensus sequence at the “splice donor site” for RNAsplicing. Therefore, the RNA splicing of fad3E-2 primary transcript(pre-mRNA) cannot be processed to create a mature RNA (mRNA).

Large scale screening of the parental lines (1904-35 and 95CB504) aswell as other Brassica napus cultivars including 2558, indicated thefad3E-2 SNP allele was 1904-35-specific and was significantly associatedwith the low ALA phenotype (R-square=0.275 for C18:3 content) using 217DH lines developed from the cross between 1904-35 and 95CB504. This1904-35 fad3E-2 SNP allele also was present in selections having <1.5%C18:3 content from a backcross population developed from the crossbetween 1904-35 and 1035R, an R line (restorer).

Example 3 Identification of a Fad3D Mutation in 1904-35 Plants

As indicated in Example 2, a 2^(nd) QTL was also identified for lowlinolenic acid content. Comparative genomics located this 2nd QTL on theN5 chromosome of Brassica napus and further identified a Fad3D candidategene on chromosome N5. The Fad3D genes from chromosome N5 of theBrassica napus genome were sequenced from 1904-35, 95CB504, and IMC201.The sequences were analyzed using BLAST and DNASTAR/Lasergene 8.0(DNASTAR, Inc). FIG. 4 shows the sequence alignment of a portion of theBnFad3D gene from 1904-35, 95CB504 and IMC201.

A deletion was identified in one of the two Fad3D isoforms from the1904-35 mutant line that was not present in 95CB504 and IMC201. Themutant type BnFad3D from 1904-35 has a deletion including a portion ofexon 1 (from position 575 to position 739). In IMC201 and 95CB504, exon1 starts at position 441 and ends at position 739. As a result of thedeletion in 1904-35, exon 1 is only 134 bp long. Therefore, it isbelieved the deletion mutation in 1904 BnFad3D induced a non-functionaltruncated protein/enzyme due to either lack of RNA splicing (truncatedprotein with 64 amino acids) or incorrect RNA splicing (truncatedprotein).

Large scale screening of the parental lines (1904-35 and 95CB504) aswell as other Brassica napus cultivars, indicated the Fad3D deletion was1904-35-specific. In addition, the Fad3D deletion was significantlyassociated with the low ALA phenotype (R-square=0.61, equal to 61%phenotypic variation on C18:3) using the parental lines and 77 DH linesdeveloped from the cross between 1904-35 and 95CB504 compared with 22%explained by BnFad3E-2 mutation in 1904-35. In order to determine therelative effect of individual Fad3 isoform on C18:3 content, 215 lineswere used from multiple populations, which carry all Fad3 isoforms, forthe multiple regression analysis. Results demonstrated that BnFad3Bexplains the largest proportion of phenotypic variation on C18:3 contentwith 26%, followed by 16% by BnFad3D, 8% by BnFad3A, and 7% by BnFad3E.

Example 4 Mutant Fad3A and Fad3B Genes

A population of B. napus IMC201 seeds was subjected to chemicalmutagenesis as set forth in Example 1. Approximately 200,000 treatedseeds were planted in standard greenhouse potting soil and placed intoenvironmentally controlled greenhouse. The plants were grown undersixteen hours of day light. At maturity, M2 seed was harvested from theplants and bulked together. The M2 generation was planted and leafsamples from the early, post-cotyledon stage of development from 8plants were pooled and DNA was extracted from leaves of these plants.The leaf harvest, pooling and DNA extraction was repeated forapproximately 32,000 plants, and resulted in approximately forty 96-wellblocks containing mutagenized B. napus IMC201 DNA. This grouping ofmutagenized DNA is referred to below as the original DNA mutagenesislibrary.

Additionally, approximately 200,000 treated seeds from the dual mutagentreatment described in Example 1 were planted in standard greenhousepotting soil and placed into environmentally controlled greenhouse. Theplants were grown under sixteen hours of day light. At maturity, M2 seedwas harvested from the plants and bulked together. This M2 generationwas planted in greenhouses and, at flowering, plants were bagged ingroups of four to facilitate cross-pollination that would occur inparallel with the majority self pollination events, and seed from thisgeneration was harvested. Genomic DNA from three seeds per plant of thisM3 generation was isolated in 96-well blocks; a collection ofmutagenized DNA from this process is referred to below as the newTilling DNA mutagenesis library.

The original DNA mutagenesis library and the new Tilling DNA mutagenesislibrary were screened to identify stop-codon containing fad3A and fad3Bmutant alleles. PCR reactions were performed using B. napus IMC201genomic DNA original mutagenesis library or new Tilling DNA mutagenesislibrary. PCR products from the original mutagenesis library wereanalyzed using temperature gradient capillary electrophoresis on aREVEAL® instrument (Transgenomics Inc.), which allows PCR reactionscontaining heterogeneous PCR products to be distinguished from reactionscontaining only homogeneous products, as would be the case if a SNPexisted in genomic DNA from chemical mutagenesis and subsequent PCRamplification. The PCR products from the new Tilling DNA mutagenesislibrary were sequenced directly using an Applied Biosystems (LifeTechnologies) 3730 DNA sequencer using the manufacturer'srecommendations.

Individual seeds representing the primary hit of each M2 plant that wasthe source genomic DNA mix for this primary mutagenesis screen weresampled and genomic DNA was isolated in order to perform the Fad3A PCRon these individuals. PCR products were sequenced and the sequences werecompared to the wild-type sequence to screen for the presence of aninduced stop codon.

The sequence comparisons indicated that a mutation had been generatedand mutant plants obtained for each of the Fad3A and Fad3B genes. Themutant Fad3A sequence is shown in SEQ ID NO: 9 and contains a mutationat position 102, changing the codon from TGG to TGA. The mutant Fad3Bsequence is shown in SEQ ID NO:10 and contains a mutation at position206, resulting in a codon change from TGG to TAG.

Example 5 DH Line Husker

A cross was made between 1904-35 (Example 1) and 95CB504, a B line(maintainer). A double haploid population was generated by collecting F₁microspores from the cross, treating the microspores with colchicine,and propagating them in vitro. Plantlets formed in vitro from themicrospores were moved to a greenhouse and inflorescences that formedwere self pollinated. Seed was harvested from the DH₁ plants at maturityand analyzed for fatty acid profile. Seeds from those plants exhibitinglow and high linolenic acid content were grown in the greenhouse. Table3 contains the fatty acid profile of a bulk sample of seeds produced byeach of 5-10 greenhouse-grown plants of a DH₁ population designatedHusker.

TABLE 3 Fatty acid profile of DH₁ population designated Husker RESCHIDPedigree n C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 C20:0 Husker-95CB504xM3B- 5 0.00 2.308 0.078 1.728 80.758 11.130 0.766 0.774 1001904-35 Husker- 95CB504xM3B- 3 0.00 3.353 0.140 2.310 78.293 11.9200.923 0.887 141 1904-35 Husker- 95CB504xM3B- 5 0.03 3.336 0.160 2.04878.186 11.984 0.858 0.938 147 1904-35 Husker- 95CB504xM3B- 5 0.03 3.6880.242 2.246 78.816 10.864 0.814 0.920 161 1904-35 Husker- 95CB504xM3B- 50.04 4.130 0.256 2.070 75.004 12.408 2.444 0.934 107 1904-35 Husker-95CB504xM3B- 5 0.02 3.363 0.175 2.080 78.211 11.661 1.161 0.891 1251904-35 Husker- 95CB504xM3B- 5 0.02 3.574 0.195 2.151 77.702 11.7671.240 0.914 138 1904-35 Husker- 95CB504xM3B- 4 0.04 4.768 0.268 1.80875.962 11.738 2.336 0.780 170 1904-35 Husker- 95CB504xM3B- 5 0.01 2.7650.153 1.900 79.270 12.533 0.738 0.718 314 1904-35 Husker- 95CB504xM3B- 50.03 4.526 0.168 2.370 75.004 11.948 2.338 0.934 323 1904-35 95CB504 90.05 3.723 0.231 2.518 78.586 9.073 2.256 1.030 TOT RESCHID Pedigree nC20:1 C20:2 C22:0 C22:1 C24:0 C24:1 SATS Husker- 95CB504xM3B- 5 1.4680.030 0.442 0.008 0.266 0.248 5.516 100 1904-35 Husker- 95CB504xM3B- 31.227 0.000 0.447 0.000 0.233 0.273 7.227 141 1904-35 Husker-95CB504xM3B- 5 1.346 0.040 0.550 0.000 0.310 0.224 7.210 147 1904-35Husker- 95CB504xM3B- 5 1.330 0.016 0.484 0.004 0.304 0.244 7.670 1611904-35 Husker- 95CB504xM3B- 5 1.474 0.030 0.598 0.006 0.364 0.242 8.140107 1904-35 Husker- 95CB504xM3B- 5 1.369 0.023 0.504 0.004 0.295 0.2467.153 125 1904-35 Husker- 95CB504xM3B- 5 1.349 0.022 0.517 0.003 0.3010.246 7.480 138 1904-35 Husker- 95CB504xM3B- 4 1.344 0.018 0.478 0.0000.232 0.224 8.108 170 1904-35 Husker- 95CB504xM3B- 5 1.150 0.038 0.3380.000 0.170 0.220 5.903 314 1904-35 Husker- 95CB504xM3B- 5 1.568 0.0340.538 0.010 0.324 0.208 8.722 323 1904-35 95CB504 9 1.446 0.040 0.5330.017 0.321 0.189 8.163

Example 6 DH Line Vest

A cross was made between 2558-35 (Example 1) and Dumpling-314, a doublehaploid B-line (maintainer) developed from a cross between IMC106RR andJetton, a known winter rapeseed variety. A double haploid population wasgenerated as described in Example 5. Seed was harvested from the DH₁plants at maturity and analyzed for fatty acid profile. Seeds from thoseplants exhibiting low linolenic acid content were grown in thegreenhouse. Table 4 contains the fatty acid profile of a bulk sample ofseeds produced by each of 10 greenhouse-grown plants of a DH₁ populationdesignated Vest.

TABLE 4 Mean fatty acid profile of Vest DH lines from tails of C18:3distribution (Vest Population N = 51) Research ID Pedigree C14:0 C16:0C16:1 C18:0 C18:1 C18:2 C18:3 C20:0 Vest-70 Dump314-05xM3B- 0.069 4.5070.223 1.983 67.152 22.456 0.653 0.778 2558-35-2 Vest-52 Dump314-05xM3B-0.060 4.001 0.308 1.844 77.279 12.080 0.680 0.876 2558-35-2 Vest-86Dump314-05xM3B- 0.065 4.406 0.264 2.266 79.352 9.008 0.745 1.0092558-35-2 Vest-60 Dump314-05xM3B- 0.072 4.685 0.273 3.143 77.323 10.1430.787 1.217 2558-35-2 Vest-87 Dump314-05xM3B- 0.082 5.083 0.346 2.31768.253 18.677 0.790 1.001 2558-35-2 Vest-75 Dump314-05xM3B- 0.101 5.0600.442 2.760 70.842 14.678 1.830 1.142 2558-35-2 Vest-69 Dump314-05xM3B-0.143 5.840 0.653 3.079 65.526 18.353 1.952 1.359 2558-35-2 Vest-71Dump314-05xM3B- 0.103 5.719 0.515 4.595 57.872 23.001 2.039 1.6732558-35-2 Vest-92 Dump314-05xM3B- 0.137 6.678 0.606 3.026 54.170 28.7862.423 1.300 2558-35-2 Vest-97 Dump314-05xM3B- 0.126 6.439 0.643 3.48354.626 27.270 2.528 1.315 2558-35-2 Dumpling-314 avg 0.051 4.405 0.2552.259 67.459 20.564 1.396 0.969 (n = 4) Research TOT ID Pedigree C20:1C20:2 C22:0 C22:1 C24:0 C24:1 SATS Vest-70 Dump314-05xM3B- 1.191 0.0660.431 0.048 0.249 0.193 8.017 2558-35-2 Vest-52 Dump314-05xM3B- 1.5620.053 0.571 0.043 0.411 0.233 7.762 2558-35-2 Vest-86 Dump314-05xM3B-1.486 0.044 0.623 0.044 0.468 0.220 8.838 2558-35-2 Vest-60Dump314-05xM3B- 1.287 0.042 0.597 0.000 0.430 0.000 10.145 2558-35-2Vest-87 Dump314-05xM3B- 1.284 0.064 0.624 0.000 0.505 0.976 9.6122558-35-2 Vest-75 Dump314-05xM3B- 1.415 0.067 0.657 0.000 0.516 0.49110.235 2558-35-2 Vest-69 Dump314-05xM3B- 1.324 0.111 0.842 0.000 0.6260.192 11.889 2558-35-2 Vest-71 Dump314-05xM3B- 1.125 0.085 1.005 0.0000.660 1.608 13.755 2558-35-2 Vest-92 Dump314-05xM3B- 1.191 0.000 0.6950.000 0.628 0.359 12.464 2558-35-2 Vest-97 Dump314-05xM3B- 1.088 0.0000.712 0.441 0.616 0.715 12.690 2558-35-2 Dumpling-314 avg 1.380 0.0620.571 0.016 0.454 0.160 8.708 (n = 4)

Example 7 Development of Hybrid Canola Producing Reduced ALA in the SeedOil

A hybrid canola line yielding seeds with an ALA content of less than1.5% was produced by introducing genes from line 1904-35 (Example 1)into a commercially grown hybrid variety, Victory® v1035. Hybrid v1035has an average oleic acid content of 65% and an ALA content of 2.8%.Plants of the line 1904-35, and the inbreds 1035R and 95CB504, wereplanted in a greenhouse. Inbred 1035R is the male parent of v1035.Inbred 95CB504 is the B line female parent of v1035. Plants of 1035R and1904-35 were cross pollinated in the greenhouse, as were 95CB504 and1904-35, as shown in Table 5.

TABLE 5 Female x Male 1035R (R-line) 1904-35 95CB504 (B-line) 1904-35

F₁ progeny from the cross of 95CB504 and 1904-35 were backcrossed to95CB504 to produce BC₁-B progeny, which were selfed (BC₁S). Plants withlow total saturates were selected from the BC₁-B selfed progeny, andbackcrossed to 95CB504 to produce BC₂-B progeny. F₁ progeny from thecross of 1035R and 1904-35 were backcrossed to 1035R to produce BC₁-Rprogeny, which were selfed. Plants with low linolenic were selected fromthe BC₁-R selfed progeny, and backcrossed to 1035R to produce BC₂-Rprogeny. Backcrossing, selection, and self-pollination of the BC-B andBC-R progeny were continued for multiple generations. The 95CB504 malesterile A line, 000A05 was converted to a low linolenic phenotype inparallel with the conversion of the 95CB504 B line. Table 6 shows themean C18:3 content of selected lines of converted BC₃S₅ generationparent lines compared unconverted 95CB504 and 1035R.

Hybrid seed was generated by hand, using BC₁S₃ generation plants of the95CB504 B line as the female parent and BC₁S₃ generation plants of the1035R R line as the male parent. The hybrid seed was grown at 5locations×4 replications in Western Canada. In the trial plot locations,some individual plants were bagged for self pollination (5 locations×2reps) and seeds harvested at maturity. The remaining plants were notbagged (5 locations×4 reps) and seeds were harvested in bulk. As such,the bulk samples had some level of out crossing with non-low linolenicfatty acid lines in adjacent plots. Seeds from the individual and bulksamples were analyzed for fatty acid content. Seeds from control plantsof line Q2, hybrid v1035 and commercial variety 46A65 were alsoharvested individually and in bulk.

Table 7 shows the fatty acid profile of the individually bagged samplesand bulked samples for hybrid 1904-35 and controls. The results indicatethat seed produced by Hybrid 1904-35 has a statistically significantdecrease in 18:3 content relative to the controls.

TABLE 6 Mean linolenic acid content of parental controls and convertedparents. ANOVA BC3S5 Populations Greenhouse ID Pedigree Mean C18:3 N09AP:Waring42 a 1035R 1.914 8 09AP:Waring43 a 95CB504 1.892 609AP:Waring30 b 95CB504xM3B-1904 1.231 20 09AP:Waring25 b95CB504xM3B-1904 1.226 20 09AP:Waring27 b 95CB504xM3B-1904 1.206 2009AP:Waring28 b 95CB504xM3B-1904 1.198 20 09AP:Waring29 b95CB504xM3B-1904 1.174 20 09AP:Waring32 b 95CB504xM3B-1904 1.165 2009AP:Waring26 b 95CB504xM3B-1904 1.163 20 09AP:Waring33 b95CB504xM3B-1904 1.146 20 09AP:Waring31 c 95CB504xM3B-1904 1.081 2009AP:Waring7 d 1035R BxM3B-1904 0.797 20 09AP:Waring18 de 1035RBxM3B-1904 0.780 20 09AP:Waring14 def 1035R BxM3B-1904 0.773 2009AP:Waring11 def 1035R BxM3B-1904 0.760 20 09AP:Waring13 def 1035RBxM3B-1904 0.755 18 09AP:Waring8 def 1035R BxM3B-1904 0.755 2009AP:Waring16 def 1035R BxM3B-1904 0.752 20 09AP:Waring21 def 1035RBxM3B-1904 0.751 19 09AP:Waring17 def 1035R BxM3B-1904 0.751 2009AP:Waring12 def 1035R BxM3B-1904 0.737 20 09AP:Waring19 def 1035RBxM3B-1904 0.728 20 09AP:Waring10 efg 1035R BxM3B-1904 0.692 2009AP:Waring9 efg 1035R BxM3B-1904 0.691 20 09AP:Waring20 efg 1035RBxM3B-1904 0.681 20 09AP:Waring15 fg 1035R BxM3B-1904 0.668 2009AP:Waring23 g 1035R BxM3B-1904 0.634 20 09AP:Waring22 g 1035RBxM3B-1904 0.632 19 09AP:Waring24 g 1035R BxM3B-1904 0.598 20 *Demonstrates significant difference between recurrent parent andbackcross selections

TABLE 7 Mean linolenic fatty acid content of converted hybrid, v1035 andcontrols. reschid mean s.e. N Student-Newman-Keuls Tests for C18_3 DataPooled for Bulked and Selfed Seed Q2 8.142 a 0.218 30 46A65 7.528 b0.098 29 V1035 2.874 c 0.170 31 1904 Conversion 1.504 d 0.118 30 DataSeparated for Bulked and Selfed Seed Q2 8.206 a 0.536 11 Q2 Bulk 8.105 a0.166 19 46A65 7.823 a 0.168 10 46A65 Bulk 7.372 a 0.107 19 V1035 3.156b 0.481 10 V1035 Bulk 2.739 b 0.108 21 1904 Conversion 1.546 c 0.123 19Bulk 1904 Conversion 1.431 c 0.250 11

Example 8

Crosses were made between selections from lines of double haploidpopulation Vest (Example 6) and reduced saturated fatty acid lines F6(1764-43-06×1975-90-14) and 06JAXB (01OB054R×15.36). The reducedsaturated fatty acid lines are described in U.S. Provisional ApplicationNo. 61/287,985, filed Dec. 18, 2009, and U.S. Provisional ApplicationNo. 61/295,049, filed Jan. 14, 2010. Double haploid populations weregenerated from these crosses as described in Example 4. Seed washarvested from the DH1 plants at maturity and analyzed for fatty acidprofile. Table 8 contains the fatty acid profile of seeds produced byeach of 1 greenhouse-grown plant from DH1 populations designated GP#1,GP#4 and GP#5 as well as 3 parental lines grown as reduced linolenic andtotal saturated fatty acid controls.

TABLE 8 Research ID Pedigree C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3C20:0 GP #1- Vest-57x(1764-43-6x1975- 0.03 2.62 0.16 1.06 77.82 14.261.03 0.46 396 90-14) GP #1-24 Vest-57x(1764-43-6x1975- 0.05 3.11 0.290.95 74.63 16.92 1.06 0.47 90-14) GP #1- Vest-57x(1764-43-6x1975- 0.043.11 0.26 1.02 75.24 16.41 1.06 0.46 181 90-14) GP #1-Vest-57x(1764-43-6x1975- 0.03 2.78 0.23 1.15 76.61 14.89 0.99 0.53 44490-14) GP #1- Vest-57x(1764-43-6x1975- 0.03 3.02 0.25 1.11 77.22 14.221.08 0.51 449 90-14) GP #1- Vest-57x(1764-43-6x1975- 0.00 2.80 0.20 1.3574.47 16.93 1.04 0.55 240 90-14) GP #1- Vest-57x(1764-43-6x1975- 0.042.59 0.19 1.54 79.41 12.02 0.97 0.63 150 90-14) GP # 4-Vest-70x(1764-43-6x1975- 0.02 2.74 0.17 1.28 75.26 16.19 1.04 0.55 2090-14) GP # 4- Vest-70x(1764-43-6x1975- 0.03 3.48 0.16 1.22 65.47 25.830.82 0.51 17 90-14) GP # 5- (01OB054RxLSAt15.36)xVest- 0.03 2.83 0.131.56 77.51 13.35 1.09 0.66 434 57-05 GP # 5-* (01OB054RxLSAt15.36)xVest-0.03 2.99 0.23 1.31 86.21 4.37 1.11 0.67 57-05 GP # 5-(01OB054RxLSAt15.36)xVest- 0.02 3.47 0.00 1.43 76.99 14.12 0.86 0.60 35157-05 GP # 5- (01OB054RxLSAt15.36)xVest- 0.03 3.16 0.18 1.52 73.74 16.871.05 0.62 334 57-05 GP # 5- (01OB054RxLSAt15.36)xVest- 0.03 2.96 0.151.61 79.04 11.59 1.08 0.70 404 57-05 GP # 5- (01OB054RxLSAt15.36)xVest-0.04 3.16 0.22 1.61 86.99 3.90 1.02 0.66 332 57-05 GP # 5-(01OB054RxLSAt15.36)xVest- 0.04 2.92 0.23 1.71 86.45 4.10 0.97 0.79 34457-05 (1764-43-6x1975-90-14) avg 0.03 2.89 0.21 1.17 70.30 18.55 2.840.61 (n = 12) Vest-57 avg (n = 10) 0.04 4.17 0.23 1.67 77.79 11.35 0.910.83 Vest-70 avg (n = 12) 0.06 4.76 0.21 1.60 65.52 23.50 0.95 0.74Research TOT ID Pedigree C20:1 C20:2 C22:0 C22:1 C24:0 C24:1 SATS GP #1-Vest-57x(1764-43-6x1975- 1.67 0.10 0.34 0.05 0.21 0.20 4.72 396 90-14)GP #1-24 Vest-57x(1764-43-6x1975- 1.60 0.07 0.33 0.04 0.17 0.30 5.0890-14) GP #1- Vest-57x(1764-43-6x1975- 1.55 0.07 0.32 0.04 0.19 0.225.15 181 90-14) GP #1- Vest-57x(1764-43-6x1975- 1.72 0.07 0.40 0.04 0.270.27 5.16 444 90-14) GP #1- Vest-57x(1764-43-6x1975- 1.68 0.08 0.34 0.030.18 0.25 5.20 449 90-14) GP #1- Vest-57x(1764-43-6x1975- 1.64 0.10 0.380.06 0.25 0.24 5.32 240 90-14) GP #1- Vest-57x(1764-43-6x1975- 1.67 0.090.38 0.03 0.22 0.23 5.39 150 90-14) GP # 4- Vest-70x(1764-43-6x1975-1.76 0.10 0.35 0.06 0.26 0.23 5.21 20 90-14) GP # 4-Vest-70x(1764-43-6x1975- 1.58 0.11 0.34 0.05 0.16 0.25 5.74 17 90-14) GP# 5- (01OB054RxLSAt15.36)xVest- 1.76 0.10 0.44 0.05 0.24 0.25 5.76 43457-05 GP # 5-* (01OB054RxLSAt15.36)xVest- 1.92 0.05 0.52 0.05 0.29 0.265.80 57-05 GP # 5- (01OB054RxLSAt15.36)xVest- 1.71 0.06 0.34 0.04 0.190.18 6.04 351 57-05 GP # 5- (01OB054RxLSAt15.36)xVest- 1.69 0.10 0.410.05 0.31 0.26 6.05 334 57-05 GP # 5- (01OB054RxLSAt15.36)xVest- 1.740.08 0.49 0.05 0.29 0.18 6.08 404 57-05 GP # 5-(01OB054RxLSAt15.36)xVest- 1.44 0.05 0.44 0.05 0.27 0.15 6.18 332 57-05GP # 5- (01OB054RxLSAt15.36)xVest- 1.76 0.07 0.48 0.04 0.24 0.20 6.19344 57-05 (1764-43-6x1975-90-14) avg 1.92 0.15 0.50 0.12 0.35 0.37 5.54(n = 12) Vest-57 avg (n = 10) 1.57 0.06 0.63 0.06 0.42 0.28 7.75 Vest-70avg (n = 12) 1.40 0.09 0.52 0.06 0.33 0.28 8.01

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A Brassica plant comprising a modified allele at a fatty aciddesaturase 3D (fad3D) locus or a fatty acid desaturase 3E (fad3E) locus,wherein said modified allele results in the production of a FAD3D orFAD3E polypeptide having reduced desaturase activity relative to acorresponding wild-type FAD3 polypeptide.